Ligand for vascular endothelial growth factor receptor

ABSTRACT

The present invention relates to compositions comprised of a peptide ligand or derivatives thereof that are capable of specific binding to the high affinity receptor-1 of vascular endothelial growth factor (VEGF) and structure similar receptors. The invention further provides a peptide ligand or derivatives thereof that are capable of inhibiting angiogenesis induced by VEGF. The present invention also provides a method for treatment or diagnosis of disease associated with angiogenesis in a patient in need of therapy comprising administering to the patient an effective amount of the pharmaceutical composition of the present invention and a pharmaceutical acceptable carrier.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/775,743, filed onFeb. 2, 2001 now U.S. Pat. No. 6,733,755.

FIELD OF THE INVENTION

The present invention relates to compositions comprised of a peptideligand or derivatives thereof (hereinafter, “Ligand”) that are capableof specifically binding to the high affinity receptor-1 of vascularendothelial growth factor (hereinafter, “VEGF”) and structure similarreceptors. The invention further describes the Ligand that is capable ofinhibiting angiogenesis induced by VEGF. The invention further describescompositions comprised of a Ligand, or its complex with a carrierassociated with a biological agent. The invention further relates to amethod of inhibiting angiogenesis by using a Ligand or its complex witha carrier alone or associated with a biological agent. The inventionfurther relates to a method for targeting of a biological agent to apredetermined compartment by associating the agent with a Ligand or itscomplex with a carrier. These compositions are well suited for use astherapeutic and diagnostic agents for the pathologies that areassociated with an increased level of VEGF receptors, and as vehiclesfor delivering biologically active and diagnostic agents to the sites inwhich VEGF receptor levels are increased.

BACKGROUND OF THE INVENTION

The growth of new blood vessels from existing endothelium (angiogenesis)is tightly controlled in healthy adults by opposing effects of positiveand negative regulations. Under certain pathological conditions,including proliferative retinopathies, rheumatiod arthritis, psoriasisand cancer, positive regulations prevail and angiogenesis contributes todisease progression (Folkman (1995) Nature Medicine 1:27–31; Achen andStacker (1998) Int. J. Exp. Pathol. 79:255–265). In cancer, the notionthat angiogenesis represents the rate limiting step of tumor growth andmetastasis, (Folkman (1971) New Engl. J. Med. 285:1182–1186) is nowsupported by considerable experimental evidence (reviewed in Aznavoorianet al. (1993) Cancer 71:1368–1383; Bidfer and Ellis (1994) Cell79:185–188; Plate and Warnke (1997) J. Neurooncol 35:365–372; de Jong etal. (1998) J. Pathol 184:44–52).

A number of angiogenic growth factors have been described to date amongwhich vascular endothelial growth factor (VEGF) appears to play a keyrole in the regulation of vasculogenesis and angiogenesis as a highlyspecific mitogen for endothelial cells (Brown et al., (1997) Control ofAngiogenesis (Goldberg and Rosen, eds) Birkhauser, Basel, pp 233–269;Martiny-Broun and Marme(1995) Current Opin. in Biotech. 6:675–680;Ferrara and Davis-Smyth (1997) Endocrine Reviews 18:4–25).

VEGF is a glycosylated, disulfide-linked homodimeric protein consistingof two 23 kD subunits. Four different monomeric isoforms of VEGF existranging in size from 121 to 206 residues in humans (VEGF₁₂₁, VEGF₁₆₅,VEGF₁₈₉ and VEGF₂₀₆). Transcripts encoding the three shorter forms aredetected in the majority of tumor cells and tumor tissue expressing VEGFgene. The isoforms result from different splicing events, and allvariants share the same 115 N-terminal as well as six C-terminalresidues and have a leader sequence to leave the cells. VEGF₁₆₅ is thedominant isoform, while VEGF₂₀₆ has so far only been identified in humanfetal liver cDNA library VEGF₁₆₅ and VEGF₁₈₉ bind heparin with highaffinity, and are sequestered to the cell surface or within theextracellular matrix bound to proteoglycans, while VEGF₁₂₁ does not bindheparin and is thus freely diffusible. Plasmin cleavage of VEGF₁₆₅generates a 110-residue long N-terminal fragment (the receptor-bindingdomain) that no longer binds heparin but is equipotent to VEGF121 in itsability to induce endothelial cell proliferation.

VEGF is expressed in embryonic tissues (Breier et al., (1992)Development (Camb.) 114:521), macrophages, proliferating epidermalkeratinocytes during wound healing (Brown et al., (1992) J. Ex. Med.176:1375–9) and may be responsible for tissue edema associated withinflammation (Ferrara and Davis-Smyth (1997) Endocrine Reviews 18:4–25).In situ hybridization studies have demonstrated high VEGF expression ina number of human tumors including glioblastoma, ovarian tumors,carcinoma, hemangioblastoma, brain neoplasms and Kaposi's sarcoma (Plateet al., (1992) Nature 359:845–848; Zebrowski et al., (1999) Ann. Surg.Oncol. 6:373–378). High levels of VEGF were also observed inhypoxia-induced angiogenesis (Shweiki et al., (1992) Nature359:843–845).

The biological function of VEGF is mediated through binding to two highaffinity receptors which are selectively expressed on endothelial cellsduring embryogenesis (Millauer et al., (1993) Cell 72:835–838) and VEGFrelated pathologies (tumor formation). VEGF receptors include the humankinase domain receptor (KDR), described in U.S. Pat. No. 5,712,380; itsmurine analog flk-1, sequenced by Mallhews (1991) Proc. Natl. Acad. Sci.USA, 88:9026–9030; U.S. Pat. No. 5,270,458 and the Fsm-like tyrosinekinase (Flt-1) (Shibuya et al., (1990) Oncogene 5:519–524). All of themare class III tyrosine kinases (Vaisman et al., 1990: J. Biol. Chem.265, 19461–19466; Kaipainen et al., (1993) J. Exp. Med. 178:2077–2088).Studies in mice have shown that the expression of KDR reaches thehighest levels during embryonic vasculogenesis and angiogenesis(Millauer et al., 1993 Cell 72:835–838). In contrast, only low levels ofmRNA for Flt-1 were found during fetal growth and moderate levels duringorganogenesis, but high levels in newborn mice (Peters et al., 1993Proc. Natl. Aca. Sci. U.S.A 90(16):7533–7). Experiments with knockoutmice deficient in either receptor revealed that KDR is essential for thedevelopment of endothelial cells, whereas Flt-1 is necessary for theorganization of embryonic vasculature (Fong et al., 1995 Dev. Dyn.203(1):80–92; Shalaby et al., 1995 Nature 376 (6535:62–6).

KDR and Flt-1, each ˜1300 amino acid residues long, are composed of 7extracellular Ig-like domains containing the ligand-binding region, asingle short membrane-spanning sequence, and an intracellular regioncontaining tyrosine kinase domains. The amino acid sequences of KDR andFlt-1 are ˜45% identical to each other. Flt-1 has the higher affinityfor VEGF (K_(D)=10–20 pM) compared to 75–125 pM for the KDR receptor.VEGF binding to KDR but not Flt-1 elicits an efficient (ED50˜0.1–1ng/ml) DNA synthetic and chemotactic endothelial cell response.Activation of Flt-1 receptor by VEGF might modulate the interaction ofendothelial cells with each other or the basement membrane on which theyreside.

The Flt-1 receptor mRNA can be spliced to generate forms encoding eitherthe full-length membrane-spanning receptor or a soluble form, denotedsFlt-1. Pure sFlt-1 retains its specific high affinity binding for VEGFand fully inhibits VEGF-stimulated endothelial cell mitogenesis bydominant negative mechanism.

Like other growth factor transmembrane tyrosine kinase receptors, VEGFreceptors presumably undergo ligand-induced dimerization, that triggerssignal transduction by promoting either autophosphorylation ortransphosphorylation specific downstream signal transduction proteinmediators.

To gain a better understanding of the biological activity of VEGF theanalysis of structure/activity relationships was performed usingsite-directed mutagenesis and epitope mapping of neutralizing monoclonalantibodies (Keyt et al., (1996) J. Biol. Chem. 271:5638–5646). Arg82,Lys84 and His86, located in a hairpin loop, were found to be criticalfor binding KDR/Flk-1, while negatively charged residues, Asp63, Glu64and Glu67, were associated with Flt-1 binding. The three-dimensionalstructure of the receptor-binding domain of VEGF (residues 8–109) showedthat these positively and negatively charged regions are distal in themonomer but are spatially close in the dimer (Wiesmann et al., (1997)Cell 91:695–704). Mutations within the KDR site had minimal effect onFit-1 binding, suggesting that receptors have different binding sites onVEGF which may serve to dimerize tyrosine kinase receptors resulting ininitiation of angiogenesis.

Domain deletion studies on Flt-1 receptor have shown that the ligandbinding function resides within the first three domains (Barleon et al.,(1997) J. Biol. Chem. 272:10382–1038; Cunningham et al., (1997) Biochim.Biophys. Res. Commun. 231 (3): 596–599), and domain 4 is required toefficiently couple ligand binding to signal transduction by means ofdirect receptor-receptor contacts (Barleon et al., (1997) J. Biol. Chem.272:10382–10388). The crystal structure of the complex between VEGF andthe second domain of Flt-1 showed domain 2 in a predominantlyhydrophobic interaction with the “poles” of VEGF dimer (Wiesmann et al.,(1997) Cell 91:695–704). Deletion experiments on KDR demonstrated thatonly domain 2 and 3 are critical for ligand binding (Fuh et al., (1998)J. Biol Chem. 1998; 273 (18):11197–204).

Endothelial cells also contain another type of VEGF receptors,Neuropilins (NP), possessing a lower mass than either VEGFR2 or VEGFR1(Gluzman-Poltorak Z., et al., (2000) J. Biol. Chem., 275(24):18040–5; WOPatent 0002/3565A2). It was subsequently found that these smaller VEGFreceptors of endothelial cells are isoform specific receptors that bindVEGF165 but not VEGF121 (Gluzman-Poltorak Z, et al., (2000) J. Biol.Chem., 275(38):29922). Unusually large amounts of these isoform-specificreceptors were found in several types of prostate and breast cancer celllines (Miao, H. Q., et al., (2000) FASEB J., 14(15):2532–9).Neuropilin-1 is likely to play an important role in the development ofthe cardiovascular system. Gene disruption studies have indicated thatnp-1 participates in embryonic vasculogenesis and angiogenesis and isinvolved in the maturation of blood vessels, since mouse embryos lackinga functional np-1 gene die because their cardiovascular system fails todevelop properly (Kawasaki, T., et al., (1999) Neurobiol. 39(4):579–89).Subsequent experiments have shown that NP-1 also serves as a receptorfor the heparin-binding form of placenta growth factor (PlGF), PlGF-2,and for VEGF-B.

In addition to its normal physiological role, VEGF receptors areassociated with numerous pathologies, including cancer, rheumatiodarthritis, diabetic retinopathy and psoriasis; development of VEGFantagonists, blocking the interaction between VEGF and its receptorswith is therefore clinically attractive. Humanized neutralizingantibodies have been shown to interact with VEGF near the KDR and Flt-ibinding sites (Kim, K. J., et al., (1993) Nature 362, 841–844; Muller,Y. A., et al., (1997) Proc. Nat. Acad. Sci. 94, 7192–7197; Muller, Y. A.et al., (1998) Structure 6:1153–1167; U.S. Pat. No. 5,855,866), andSELEX-derived RNA molecules (Jellinek, D. et al., (1994) Biochemistry33:10450–10456; U.S. Pat. No. 5,859,228), that target VEGF, suppresstumor growth that is dependent on vascularization of adjacent normaltissue (Plate, K. H. et al., (1994) Brain-Pathol., 4:207–218). Anti KDRmonoclonal antibodies inhibited VEGF induced signaling and demonstratedhigh anti-tumor activity (Witte et al., (1998) Cancer & Metast. Reviews17:155–161; U.S. Pat. No. 5,840,301). Soluble Fit receptor (U.S. Pat.No. 5,861,484), fragments of VEGF (U.S. Pat. No. 5,240,848) have beenshown to inhibit factor/receptor interaction and angiogenesis in vivo.Anti VEGF antisense oligonucleotide was designed to inhibit VEGFexpression and VEGF induced neovascularization (U.S. Pat. No.5,641,756).

The present invention describes a new ligand for VEGF receptor-1 andsimilar receptors with is useful for targeted delivery of therapeutics.Such treatment should be as devoid as possible of undesired side effectssuch as those associated with conventional chemotherapy and some of theexperimental biotherapies.

SUMMARY OF THE INVENTION

The present invention provides a pharmaceutical composition comprisingat least one peptide or derivative thereof, wherein said polypeptide orderivative thereof is capable of specific binding with the high affinityVEGF receptor-1 or a derivative of the VEGF receptor-1 and structuralsimilar receptors. Such a polypeptide is herein referred to as Ligand.

In one embodiment, the present invention provides a peptide orderivative thereof that is capable of specific binding with VEGFreceptors.

In another embodiment, a pharmaceutical composition of the presentinvention comprises at least one peptide or derivative thereof, whereinsaid peptide or derivative thereof is capable of specific binding withthe high affinity VEGF receptor-1 or a derivative of the VEGF receptor-1and structural similar receptors. Such a composition may furthercomprise the ability to modulate the interaction of VEGF with its highaffinity VEGF receptor and modulates biological effects mediated bybinding.

A preferred peptide of the present invention provides the amino acidsequence of Asn-Gly-Tyr-Glu-Ile-Glu-Trp-Tyr-Ser-Trp-Val-Thr-His-Gly-Met-Tyr (SEQ ID NO: 1) wherein the peptide is a ligand of VEGF receptor-1and inhibits binding of VEGF to this receptor.

The present further provides a pharmaceutical composition, wherein thepolypeptide or derivative thereof comprise the amino acid sequence ofSEQ ID NOs: 1; 2; 3; 4; 5; or 7.

A preferred peptide, variant or derivative of peptide of the presentinvention has the sequence Asn-X-X-Glu-Ile-Glu-X-X-X-Trp-X-X-X-X-X-Tyr(SEQ ID NO: 7) (also abbreviated in single letter amino acid code asNXXEIEXXXWXXXXXY), where X is any amino acid.

Another embodiment of the present invention provides at least onepeptide compound having the motif of SEQ ID NO: 8:

-   Y₁-X-X-Y₂-Y₃-Y₄-X-X-X-Y₅-X-X-X-X-X-Y₆, where-   Y₁ is Asn or Gln-   Y₂ is negatively charged amino acid comprising of Glu or Asp-   Y₃ is Ile, Leu, Val or Met-   Y₄ is negatively charged amino acid comprising of Glu or Asp-   Y₅ is aromatic amino acid comprising of Trp, Phe, Tyr or His-   Y₆ is aromatic amino acid comprising of Tyr, Trp, Phe or His-   X is any amino acid,-   or a substitution variant, addition variant or other chemical    derivative thereof.

The present invention also provides analogs of the pharmaceuticalcomposition which can comprise in its molecular structure residues beingderivatives of compounds other than amino acids, referenced herein as“peptide mimetics” or “peptidomimetics”. Other analogs of peptide Ligandare compounds having changed topology of its chain, in particularnonlinear compounds, which have chemical bonds that close cycle orcycles in the molecule and constrain its structure.

The peptide Ligand of the present invention can be made by usingwell-known methods including recombinant methods and chemical synthesis.Recombinant methods of producing a peptide through the introduction of avector including nucleic acid encoding the peptide into a suitable hostcell is well known in the art, such as is described in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2d Ed, Vols. 1 to 8, Cold SpringHarbor, N.Y. (1989), which is herein incorporated by reference. A linearsequence is synthesized, for example, by the solid phase peptidesynthesis of Merrifield et al., J. Am. Chem. Soc., 85:2149 (1964), whichis incorporated herein by reference). Alternatively, a peptide of thepresent invention can be synthesized using standard solution methodswell known in the art (see, for example, Bodanszky, M., Principles ofPeptide Synthesis (Springer-Verlag, 1984)), which is herein incorporatedby reference). Newly synthesized peptides can be purified, for example,by high performance liquid chromatography (HPLC), and can becharacterized using, for example, mass spectrometry or amino acidsequence analysis. Although a purity of greater than 95 percent for thesynthesized peptide is preferred, lower purity may be acceptable. Theanalogs of the peptide Ligand can be peptides with altered sequencecomprising another selection of L-α-amino acid residues, D-α-amino acidresidues, non-α-amino acid residues.

Therefore, in another embodiment, the pharmaceutical composition of thepresent invention provides that derivatives of SEQ ID NO: 1 comprise ofoligopeptides, chemical derivatives or peptidomimetic that are capableof specific binding with the high affinity VEGF receptor-1 or aderivative of the VEGF receptor-1.

In another embodiment of the invention the Ligand is associated with abiological agent. Such an association can be achieved by chemical,genetic or physical linking of the Ligand and the biological agent, orby mixing the above components, or by their co-administration.

The peptide, derivative or peptidomimetic of this invention has one ormore of the following activities:

The present invention provides a pharmaceutical composition comprisingat least one peptide or derivative thereof, wherein said polypeptide orderivative thereof is capable of specific binding with the high affinityVEGF receptor-1 or a derivative of the VEGF receptor-1 and structuralsimilar receptors wherein the polypeptide or derivative thereofcomprises at least about 20% of the biological activity of thepolypeptide or derivative thereof SEQ ID NO: 3.

Still further, the present invention provides a pharmaceuticalcomposition comprising at least one peptide or derivative thereof,wherein the polypeptide or derivative thereof is capable of specificbinding with the high affinity VEGF receptor-1 or a derivative of theVEGF receptor-1 and structural similar receptors wherein the polypeptideor derivative thereof comprises binding activity such that thepolypeptide or derivative thereof competes with the labeled polypeptidein SEQ ID NO: 4 for binding to the VEGF receptor-1.

Yet still further, the present invention provides a pharmaceuticalcomposition comprising at least one peptide or derivative thereof,wherein the polypeptide or derivative thereof is capable of specificbinding with the high affinity VEGF receptor-1 or a derivative of theVEGF receptor-1 and structural similar receptors, wherein thepolypeptide or derivative thereof comprises at least about 20% of thebiological activity of the peptide or derivative thereof SEQ ID NO: 3and binding activity such that the peptide or derivative thereofcompetes with the labeled peptide in SEQ ID NO: 4 for binding to theVEGF receptor-1.

The biological activity of the present pharmaceutical composition ismeasured but is not limited to using in vitro bioassays comprising VEGFreceptor-1 binding assay, endothelial tube formation on, MATRIGEL™extracellular matrix proteins or endothelial cell mitogenic assay.

The invention further includes a pharmaceutical composition comprisingone or more Ligands in association with a carrier. In another embodimentof the invention the Ligand is associated with a protein, polymer or anyother carrier to improve the ability of the Ligand to interact with VEGFreceptor and/or to improve pharmacological properties of the Ligand suchas pharmacokinetics, stability and biodistribution. The association ofthe Ligand and the carrier can be achieved by chemical, genetic orphysical linking of the Ligand and the carrier, or by mixing the abovecomponents, or by their co-administration.

In another yet embodiment of the invention the Ligand associated with acarrier is further associated with a biological agent which is achievedby chemical, genetic or physical linking of the Ligand—carriercomposition described in the previous embodiment of the invention and abiological agent. The invention further provides a pharmaceuticalcomposition comprising one or more Ligands or their complex with acarrier in association with a biological agent.

Moreover, the present invention extends to pharmaceutical compositionsfor diagnostics and treatment of diseases, where such diseases areassociated with angiogenesis.

The invention is further directed to a pharmaceutical composition usefulfor diagnostics or treatment of angiogenesis associated diseases,comprising (a) any of the above peptides, variants or chemicalderivatives including, but not limited to peptidorimetics and (b) apharmaceutically acceptable carrier or excipient, either chemicallyconjugated or physically associated with a ligand.

The invention is further directed to a pharmaceutical composition usefulfor diagnostics or treatment of angiogenesis associated diseases,comprising any of the above peptides, variants or chemical derivativesincluding a peptidomimetic conjugated chemically or genetically fused toa therapeutic agent.

Also provided is a method for inhibition of VEGF-induced cellproliferation or angiogenesis in a subject having a disease or conditionassociated with undesired angiogenesis, comprising administering to thesubject an effective amount of a pharmaceutical composition as describedabove.

In yet another embodiment of the invention the Ligand is associated witha modified biological agent, which is achieved by chemical linking ofthe Ligand to the modified biological agent, such an associate being apro-drug and revealing no activity of the biological agent. The activebiological agent is released from such an associate upon action ofchemical or enzymatic reaction in the body.

The present invention also provides a method of treating a diseaseassociated with angiogenesis in a patient in need of such therapycomprising administering to said patient an effective amount of saidpharmaceutical composition of claim 1 and a pharmaceutical acceptablecarrier.

In any of the foregoing methods, the disease or condition being treatedmay be primary tumor growth, tumor invasion or metastasis,atherosclerosis, post-balloon angioplasty vascular restenosis, neointimaformation following vascular trauma, vascular graft restenosis, fibrosisassociated with a chronic inflammatory condition, lung fibrosis,chemotherapy-induced fibrosis, wound healing with scarring and fibrosis,psoriasis, deep venous thrombosis, or another disease or condition inwhich angiogenesis is involved.

These and other aspects of the present invention will be betterappreciated by reference to the following Sequence Listings and DetailedDescription.

Sequence Listings SEQ ID NO: Sequence 1H-Asn-Gly-Tyr-Glu-Ile-Glu-Trp-Tyr-Ser-Trp-Val-Thr-His-Gly-Met-Tyr-NH₂ 2H-Cys-Asn-Gly-Tyr-Glu-Ile-Glu-Trp-Tyr-Ser-Trp-Val-Thr-His-Gly-Met-Tyr-NH₂3Ac-Cys-Asn-Gly-Tyr-Glu-Ile-Glu-Trp-Tyr-Ser-Trp-Val-Thr-His-Gly-Met-Tyr-NH₂4Fam-Asn-Gly-Tyr-Glu-Ile-Glu-Trp-Tyr-Ser-Trp-Val-Thr-His-Gly-Met-Tyr-NH₂5Fam-Glu-Glu-Glu-Asn-Gly-Tyr-Glu-Ile-Glu-Trp-Tyr-Ser-Trp-Val-Thr-His-Gly-Met-Tyr-NH₂ 6Fam-Asn-Gly-Tyr-Ile-Glu-Trp-Tyr-Ser-Trp-Val-Thr-His-Gly-Met-Tyr-NH₂ 7Asn-X-X-Glu-Ile-Glu-X-X-X-Trp-X-X-X-X-X-Tyr 8Y₁-X-X-Y₂-Y₃-Y₄-X-X-X-Y₅-X-X-X-X-X-Y₆

Abbreviations

-   Ac—acetyl,-   Fam—fluorescein-5-carbonyl,-   X—any amino acid,-   Y₁ is Asn or Gln,-   Y₂ is negatively charged amino acid comprising of Glu or Asp,-   Y₃ is Ile, Leu, Val or Met-   Y₄ is negatively charged amino acid comprising of Glu or Asp-   Y₅ is aromatic amino acid comprising of Trp, Phe, Tyr or His-   Y₆ is aromatic amino acid comprising of Tyr, Trp, Phe or His

Listing of conjugates CONJUGATE NO.: CONJUGATE 1 peroxidase-peptide(SEQID NO: 4) 2 PEG 1500-peptide(SEQ ID NO: 4) 3polylysine-PEG1500-peptide(SEQ ID NO: 4) 4 PEI-PEG-peptide(SEQ ID NO: 4)5 paclitaxel-PEG-peptide(SEQ ID NO: 4) 6 F127-peptide(SEQ ID NO: 4) 7paclitaxel-polyglutamic acid-peptide (SEQ ID NO: 1) 8 paclitaxel-peptide(SEQ ID NO: 1): 9 Peptide (SEQ ID NO: 3)Leucyl-doxorubicin

DETAILED DESCRIPTION OF THE INVENTION

I. The Ligand

The present invention provides a pharmaceutical composition comprisingat least one peptide, or derivatives thereof, which is capable ofspecific binding with VEGF receptors, such a peptide or its derivativereferenced herein as a “Ligand”. Since the Ligand is capable of specificbinding with the receptors, it is also able to modulate VEGF mediatedangiogenesis in endothelial cells and tissues. Therefore, presentinvention also provides pharmaceutical compositions in which the Ligandis used as a targeting moiety to improve the delivery of a biologicalagent used for therapeutic or diagnostic purpose.

In one preferred embodiment, the invention provides a Ligand of VEGFreceptors being peptide, or derivative thereof. The preparation of thepeptides or derivatives thereof according to the invention is effectedby means of one of the known organic chemical methods for peptidesynthesis or with the aid of recombinant DNA techniques.

II. Chemical Peptide Synthesis

The organic chemical methods for peptide synthesis are considered toinclude the coupling of the required amino acids by means of acondensation reaction, either in homogeneous phase or with the aid of asolid phase. The condensation reaction can be carried out as follows:

-   a) condensation of a compound (amino acid, peptide) with a free    carboxyl group and protected other reactive groups with a compound    (amino acid, peptide) with a free amino group and protected other    reactive groups, in the presence of a condensation agent;-   b) condensation of a compound (amino acid, peptide) with an    activated carboxyl group and free or protected other reaction groups    with a compound (amino acid, peptide) with a free amino group and    free or protected other reactive groups.

Activation of the carboxyl group can take place, inter alia, byconverting the carboxyl group to an acid halide, azide, anhydride,imidazolide or an activated ester, such as the N-hydroxy-succinimide,N-hydroxy-benzotriazole or p-nitrophenyl ester. The most common methodsfor the above condensation reactions are: the carbodiimide method, theazide method, the mixed anhydride method and the method using activatedesters, such as described in The Peptides, Analysis, Synthesis, BiologyVol. 1–3 (Ed. Gross, E. and Meienhofer, J.) 1979, 1980, 1981 (AcademicPress, Inc.). A particularly useful method is Castro type method usingbenzotriazole-1-yl-oxy-uronium, or -phoshponium esters, eg. PyBOP(benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate) (Martinez, J. et al. (1988) J. Med. Chem. 28,1874).

Preparation of suitable peptides according to the invention using the“solid phase” is for instance described in (1963) J. Amer. Chem. Soc.85:2149 and ((1990) Int. J. Peptide Protein Res. 35:161–214. Thecoupling of the amino acids of the peptide to be prepared usually startsfrom the carboxyl end side. For this method a solid phase is needed onwhich there are reactive groups or on which such groups can beintroduced. This can be, for example, a copolymer of benzene anddivinylbenzene with reactive chloromethyl groups, or a polymeric solidphase rendered reactive with hydroxymethyl or amine function.

After synthesis of the desired amino acid sequence, detaching of thepeptide from the resin follows, for example, hydrogen fluoride withtrifluoromethanesulphonic acid or with methanesulphonic acid dissolvedin trifluoroacetic acid. The peptide can also be removed from thecarrier by transesterification with a lower alcohol, preferably methanolor ethanol, in which case a lower alkyl ester of the peptide is formeddirectly. Likewise, splitting with the aid of ammonia gives the amide ofa peptide according to the invention.

A particularly suitable solid phase is, for example, the Rink Amideresin (4-(2′,4′-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy-copolystrene-1% divinylbenzene resin), described by Rink (1987)Tetrahedron Lett., 28:3787. After synthesis, the peptide can be splitfrom the solid phase under mild conditions using trifluoroacetic acidproducing a carboxyamide derivative.

The reactive groups, which may not participate in the condensationreaction, are, as stated, effectively protected by groups, which can beremoved again very easily by hydrolysis with the aid of acid, base orreduction. Thus, a carboxyl group can be effectively protected by, forexample, esterification with methanol, ethanol, tertiary butanol, benzylalcohol or p-nitrobenzyl alcohol and amines linked to solid support.

Groups which can effectively protect an amino group are theethoxycarbonyl, benzyloxycarbonyl, t-butoxy-carbonyl (t-boc) orp-methoxy-benzyloxycarbonyl group, or an acid group derived from asulphonic acid, such as the benzene-sulphonyl or p-toluenesulphonylgroup, but other groups can also be used, such as substituted orunsubstituted aryl or aralkyl groups, for example benzyl andtriphenylmethyl, or groups such as orthonitrophenyl-sulphenyl and2-benzoyl-1-methyl-vinyl. A particularly suitable α-amino-protectivegroup is, for example, the base-sensitive 9-fluorenyl-methoxycarbonyl(Fmoc) group (Carpino & Han (1970) J. Amer. Chem. Soc. 92, 5748). A moreextensive account of possible protecting groups can be found in ThePeptides, Analysis, Synthesis, Biology, Vol.1–9 (Eds. Gross, Udenfriendand Meienhofer) 1979–1987 (Academic Press, Inc.).

It is necessary also to protect the ε-amino group of lysine andadvisable for the guanidine group of arginine. Customary protectivegroups in this connection are a Boc group for lysine and a Pmc, Pms,Mbs, or Mtr group for arginine. The protective groups can be split offby various conventional methods, depending on the nature of theparticular group, for example with the aid of trifluoroacetic acid or bymild reduction, for example with hydrogen and a catalyst, such aspalladium, or with HBr in glacial acetic acid.

III. Biosynthetically Made Peptide

The peptides of the present invention are prepared by any technique,including by well-known recombinant methods. The above techniques aremore fully described in laboratory manuals such as “Molecular Cloning: ALaboratory Manual”, Second Edition by Sambrook et al., Cold SpringHarbor Press, 1989; “Current Protocols in Molecular Biology”, VolumesI–III, Ausubel, R. M., ed., 1994; “Cell Biology: A Laboratory Handbook”,Volumes I–III, J. E. Celis, ed., 1994; “Current Protocols inImmunology”, Volumes I–III, Coligan, J. E., ed., 1994; “OligonucleotideSynthesis”, M. J. Gait ed., 1984; “Nucleic Acid Hybridization”, B. D.Hames & S. J. Higgins eds., 1985; “Transcription And Translation”, B. D.Hames & S. J. Higgins, eds., 1984; “Animal Cell Culture”, R. I.Freshney, ed., 1986; “Immnobilized Cells And Enzymes”, IRL Press, 1986;B. Perbal, “A Practical Guide To Molecular Cloning”, 1984.

DNAs encoding the peptides of the invention can be prepared by a varietyof methods known in the art. These methods include, but are not limitedto, chemical synthesis by any of the methods described in Engels et al.,(1989) Agnew. Chem. Int. Ed. Engl., 28:716–734, the entire disclosure ofwhich is incorporated herein by reference, such as the triester,phosphite, phosphoramidite and H-phosphonate methods. In one embodiment,codons preferred by the expression host cell are used in the design ofthe ligand encoding DNA.

One example of a method of producing the ligand peptide usingrecombinant DNA techniques entails the steps of (1) syntheticallygenerating DNA oligonucleotide encoding peptide sequence, appropriatedlinkers and restriction sites coding sequences (2) inserting the DNAinto an appropriate vector such as an expression vector, (3) insertingthe gene containing vector into a microorganism or other expressionsystem capable of expressing the inhibitor gene, and (7) isolating therecombinantly produced peptides.

Those skilled in the art will recognize that the peptides of the presentinvention may also be expressed in various cell systems, bothprokaryotic and eukaryotic, all of which are within the scope of thepresent invention. The appropriate vectors include viral, bacterial andeukaryotic expression vectors. A nucleic acid molecule, such as DNA, issaid to be “capable of expressing” a polypeptide if it containsnucleotide sequences which contain transcriptional and translationalregulatory information and such sequences are “operably linked” tonucleotide sequences which encode the polypeptide. The precise nature ofthe regulatory regions needed for gene sequence expression may vary fromorganism to organism, but shall in general include a promoter regionwhich, in prokaryotes, contains both the promoter (which directs theinitiation of RNA transcription) as well as the DNA sequences which,when transcribed into RNA, will signal synthesis initiation. Suchregions will normally include those 5′-non-coding sequences involvedwith initiation of transcription and translation, such as the TATA box,capping sequence, CAAT sequence, and the like.

For example, the entire coding sequence of the ligand peptide may becombined with one or more of the following in an appropriate expressionvector to allow for such expression: (1) an exogenous promoter sequence(2) a ribosome binding site (3) carrier protein (4) a polyadenylationsignal (4) a secretion signal. Modifications can be made in the5′-untranslated and 3′-untranslated sequences to improve expression in aprokaryotic or eukaryotic cell; or codons may be modified such thatwhile they encode an identical amino acid, that codon may be a preferredcodon in the chosen expression system. The use of such preferred codonsis described in, for example, Grantham et al., (1981) Nuc. Acids Res.,9:43–74 and Lathe, (1985) J. Mol. Biol., 183:1–12, hereby incorporatedby reference herein in their entirety. Moreover, once cloned into anappropriate vector, the DNA can be altered in numerous ways as describedabove to produce functionally equivalent variants thereof.

In another embodiment, the peptide of present invention can be expressedas fusion proteins in which the peptides of invention are fused at itsN-terminus or its C-terminus, or at both termini, to one or more ofpeptide copies. In a preferred embodiment, the fusion protein isspecifically cleavable such that at least a substantial portion of thepeptide sequence can be proteolytically cleaved away from the fusionprotein to yield the desired polypeptide. The fusion proteins of theinvention can be designed with cleavage sites recognized by chemical orenzymatic proteases. In one embodiment, the fusion protein is designedwith a unique cleavage site (or sites) for removal of the ligand peptidesequence, i.e. the fusion protein is designed such that a given protease(or proteases) cleaves away the ligand peptide sequence but does notcleave at any site within the sequence of the desired protein, avoidingfragmentation of the desired protein. In another embodiment, thecleavage site (or sites) at the fusion joint (or joints) is designedsuch that cleavage of the fusion protein with a given enzyme liberatesthe authentic, intact sequence of the desired protein from the remainderof the fusion protein sequence. The pTrcHisA vector (Invitrogen) andother like can be used to obtain high-level, regulated transcriptionfrom the trc promoter for enhanced translation efficiency of fusionprotein in E. coli. The peptides of invention can be expressed fused toan N-terminal nickel-binding poly-histidine tail for one-steppurification using metal affinity resins. The enterokinase cleavagerecognition site in the fusion protein allows for subsequent removal ofthe N-terminal histidine fusion protein from the purified recombinantprotein. The ligand fusion protein can be produced using appropriatedcarrier protein, for example, β-galactosidase, green fluorescentprotein, luciferase, dehydrofolate reductase, thireodoxin, protein AStaphylococcus aureus and glutathione S-transferase. These examples are,of course, intended to be illustrative rather than limiting.

The peptides of present invention can be synthesized as a fusion proteinwith a virus coat protein and expressed on the surface of virusparticle, for example bacteriophage M13, T7, T4 and lambda (λ), λgt10,λgt11 and the like; adenovirus, retrovirus and pMAM-neo, pKRC and thelike.

In general, prokaryote expression vectors contain replication andcontrol sequences, which are derived from species compatible with thehost cell. The vector ordinarily carries a replication site, as well assequences that encode proteins capable of providing phenotypic selectionin transformed cells. For example, vectors include pBR322 (ATCC No.37,017), phGH107 (ATCC No. 40,011), pBO475, pS0132, pRIT5, any vector inthe pRIT20 or pRIT30 series (Nilsson and Abrahmsen, Meth. Enzymol., 185:144–161 (1990)), pRIT2T, pKK233-2, pDR540, pPL-lambda, pQE70, pQE60,pQE-9 (Qiagen), pBS, phagescript, psiX174, pBluescript SK, pBsKS, pNH8a,pNH16a, pNH18a, pNH46a (Stratagene); pTRC99A, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, pXT1, pSG(Stratagene) pSVK3, PBPV, pMSG, pSVL (Pharmacia) are suitable forexpression in prokaryotic hosts. Such plasmids are, for example,disclosed by Sambrook (cf. “Molecular Cloning: A Laboratory Manual”,second edition, edited by Sambrook, Fritsch, & Maniatis, Cold SpringHarbor Laboratory, (1989)). Bacillus plasmids include pC194, pC221,pT127, and the like. Such plasmids are disclosed by Gryczan (In: TheMolecular Biology of the Bacilli, Academic Press, NY (1982), pp.307–329). Suitable Streptomyces plasmids include plJ101 (Kendall et al.,(1987) J. Bacteriol. 169:4177–4183, and streptomyces bacteriophages suchas .phi.C31 (Chater et al., In: Sixth International Symposium onActinomycetales Biology, Akademiai Kaido, Budapest, Hungary (1986), pp.45–54). Pseudomonas plasmids are reviewed by John et al. ((1986) Rev.Infect. Dis. 8:693–704), and Izaki ((1978) Jpn. J. Bacteriol.33:729–742).

Prokaryotic host cells containing the expression vectors of the presentinvention include E. coli K12 strain 294 (ATCC NO. 31446), E coli strainJM101 (Messing et al., Nucl. Acid Res., 9: 309 (1981)), E. coli strainB, E. coli strain .sub.chi. 1776 (ATCC No. 31537), E. coli c600(Appleyard, (1954) Genetics, 39: 440), E. coli W3110 (F-, gamma-,prototrophic, ATCC No. 27325), E. coli strain 27C7 (W3110, tonA, phoAE15, (argF-lac)169, ptr3, degP41, ompT, kan.sup.r) (U.S. Pat. No.5,288,931, ATCC No. 55,244), Bacillus subtilis, Salmonella typhimurium,Serratia marcesans and Pseudomonas species. For example, E. coli K12strain MM 294 (ATCC No. 31,446) is particularly useful. Other microbialstrains that may be used include E. coli strains such as E. coli B andE. coli X1776 (ATCC No. 31,537). These examples are, of course, intendedto be illustrative rather than limiting.

To express of peptides of the invention (or a functional derivativethereof) in a prokaryotic cell, it is necessary to operably link thepeptide-encoding sequence to a functional prokaryotic promoter. Suchpromoters may be either constitutive or, more preferably, regulatable(i.e., inducible or derepressible). Examples of constitutive promotersinclude the int promoter of bacteriophage lambda., the bla promoter ofthe .beta.-lactamase gene sequence of pBR322, and the CAT promoter ofthe chloramphenicol acetyl transferase gene sequence of pPR325, and thelike. Examples of inducible prokaryotic promoters include the majorright and left promoters of bacteriophage lambda. (P.sub.L and P.sub.R),the trp, recA, .lambda.acZ, .lambda.acl, and gal promoters of E. coli,the .alpha.-amylase (Ulmanen et al., (1985) J. Bacteriol. 162:176–182)and the .zeta.-28-specific promoters of B. subtilis (Gilman et al.,(1984) Gene sequence 32:11–20), the promoters of the bacteriophages ofBacillus (Gryczan, In: The Molecular Biology of the Bacilli, AcademicPress, Inc., NY (1982)), and Streptomyces promoters (Ward et al., (1986)Mol. Gen. Genet. 203:468–478). The most commonly used in recombinant DNAconstruction promotors include the P-lactamase (penicillinase) andlactose promoter systems (Chang et al., (1978) Nature, 375:615; Itakuraet al., (1977) Science, 198, 1056; Goeddel et al., (1979) Nature, 281,544) and a tryptophan (trp) promoter system (Goeddel et al., (1980)Nucleic Acids Res., 8:4057; EPO Appl. Publ. No. 0036,776). While theseare the most commonly used, other microbial promoters have beendiscovered and utilized, and details concerning their nucleotidesequences have been published, enabling a skilled worker to ligate themfunctionally with plasmid vectors (see, e., Siebenlist et al., (1980)Cell, 20, 269.

Proper expression in a prokaryotic cell also requires the presence of aribosome binding site upstream of the gene sequence-encoding sequence.Such ribosome binding sites are disclosed, for example, by Gold et al.((1981) Ann. Rev. Microbiol. 35:365–404). The ribosome binding site andother sequences required for translation initiation are operably linkedto the nucleic acid molecule encoding peptides of invention. Translationin bacterial system is initiated at the codon with encode the firstmethionine. For this reason, it is preferable to include the ATG codonin peptide sequence and to ensure that the linkage between a promoterand a DNA sequence that encodes a peptide does not contain anyintervening codons that are capable of encoding a methionine.

In addition to prokaryotes, eukaryotic organisms, such as yeast, orcells derived from multicellular organisms can be used as host cells.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among eukaryotic microorganisms, although a number of other strainsare commonly available. For expression in Saccharomyces, the plasmidYRp7, for example (Stinchcomb et al., (1979) Nature 282:39; Kingsman etal., (1979) Gene 7:141; Tschemper et al., (1980) Gene 10:157), iscommonly used. This plasmid already contains the trp1 gene that providesa selection marker for a mutant strain of yeast lacking the ability togrow in tryptophan, for example, ATCC No. 44,076 or PEP4-1 (Jones,(1977) Genetics, 85, 12). The presence of the trp1 lesion as acharacteristic of the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Suitable promoting sequences in yeast vectors include thepromoters for 3-hosphoglycerate kinase (Hitzeman et al., (1980) J. Biol.Chem. 255:2073) or other glycolytic enzymes (Hess et al., (1968) J. Adv.Enzyme Reg. 7:149; Holland et al., (1978) Biochemistry 17:4900), such asenolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In constructing suitableexpression plasmids, the termination sequences associated with thesegenes are also ligated into the expression vector 3′ of the sequencedesired to be expressed to provide polyadenylation of the mRNA andtermination. Other promoters, which have the additional advantage oftranscription controlled by growth conditions, are the promoter regionfor alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism, and theaforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization. Any plasmid vectorcontaining yeast-compatible promoter, origin of replication andtermination sequences is suitable.

In addition, plant cells are also available as hosts, and controlsequences compatible with plant cells are available, such as thecauliflower mosaic virus 35S and 19S, and nopaline synthase promoter andpolyadenylation signal sequences. Another preferred host is an insectcell, for example the Drosophila larvae. Using insect cells as hosts,the Drosophila alcohol dehydrogenase promoter can be used. Rubin, (1988)Science 240:1453–1459.

However, peptides of present invention can be expressed in vertebratahost cells. The propagation of vertebrate cells in culture (tissueculture) has become a routine procedure in recent years (Tissue Culture,Academic Press, Kruse and Patterson, editors (1973). Examples of usefulmammalian host cells include monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., (1977) J. GenVirol., 36: 59); baby hamster kidney cells (BHK, ATCC CCL 10); Chinesehamster ovary cells/-DHFR (CHO, Urlaub and Chasin, (1980) Proc. Natl.Acad. Sci. USA, 77:4216); mouse sertoli cells (TM4, Mather, (1980) Biol.Reprod., 23: 243–251); monkey kidney cells (CV1 ATCC CCL 70); Africangreen monkey kidney cells (VERO-76, ATCC CRL-1587); human cervicalcarcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells(W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammarytumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., (1982) AnnalsN.Y. Acad. Sci., 383: 44–68); MRC 5 cells; FS4 cells; and a humanhepatoma cell line (Hep G2). For expression in mammalian host cells,useful vectors include, but not limited vectors derived from SV40,vectors derived from cytomegalovirus such as the pRK vectors, includingpRK5 and pRK7 (Suva et al., (1987) Science, 237:893–896, EP 307,247(Mar. 15, 1989), EP 278,776 (Aug. 17, 1988)) vectors derived fromvaccinia viruses or other pox viruses, and retroviral vectors such asvectors derived from Moloney's murine leukemia virus (MoMLV).

The expression of peptides of invention in eukaryotic hosts requires theuse of eukaryotic regulatory regions. Such regions will, in general,include a promoter region sufficient to direct the initiation of RNAsynthesis. Preferred eukaryotic promoters include, for example, thepromoter of the mouse metallothionein I gene sequence (Hamer et al.,(1982) J. Mol. Appl. Gen. 1:273–288); the TK promoter of Herpes virus(McKnight, (1982) Cell 31:355–365); the SV40 early promoter (Benoist etal., (1981) Nature (London) 290:304–310); the yeast gal4 gene sequencepromoter (Johnston et al., (1982) Proc. Natl. Acad. Sci. (USA)79:6971–6975; Silver et al., (1984) Proc. Natl. Acad. Sci. (USA)81:5951–5955). An origin of replication may be provided either byconstruction of the vector to include an exogenous origin, such as maybe derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV)source, or may be provided by the host cell chromosomal replicationmechanism. If the vector is integrated into the host cell chromosome,the latter is often sufficient. Satisfactory amounts of protein areproduced by cell cultures; however, refinements, using a secondarycoding sequence, serve to enhance production levels even further. Onesecondary coding sequence comprises dihydrofolate reductase (DHFR thatis affected by an externally controlled parameter, such as methotrexate(MTX), thus permitting control of expression by control of themethotrexate concentration (Urlaub and Chasin, (1980) Proc. Natl. Acad,Sci. (USA) 77, 4216).

Optionally, the DNA encoding peptides of invention is operably linked toa secretory leader sequence resulting in secretion of the expressionproduct by the host cell into the culture medium. Examples of secretoryleader sequences include stII, ecotin, lamB, herpes GD, lpp, alkalinephsophatase, invertase, and alpha factor. Also suitable for use hereinis the 36 amino acid leader sequence of protein A (Abrahmsen et al.,(1985) EMBO J., 4: 3901).

Once the vector or nucleic acid molecule containing the construct(s) hasbeen prepared for expression, the DNA construct(s) may be introducedinto an appropriate host cell by any of a variety of suitable means,i.e., transformation, transfection, conjugation, protoplast fusion,electroporation, particle gun technology, lipofection, calcium phosphateprecipitation, direct microinjection, DEAE-dextran transfection, and thelike. The most effective method for transfection of eukaryotic celllines with plasmid DNA varies with the given cell type. After theintroduction of the vector, recipient cells are grown in a selectivemedium, which selects for the growth of vector-containing cells.Expression of the cloned gene molecule(s) results in the production ofpeptides of invention. This can take place in the transformed cells assuch, or following the induction of these cells to differentiate (forexample, by administration of bromodeoxyuracil to neuroblastoma cells orthe like). A variety of incubation conditions can be used to form thepeptide of the present invention. The most preferred conditions arethose which mimic physiological conditions.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄.precipitation and electroporation. Successfultransfection is generally recognized when any indication of theoperation of this vector occurs within the host cell.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride, as described in section 1.82 of Sambrook etal., Molecular Cloning (2nd ed.), Cold Spring Harbor Laboratory, NewYork (1989), is generally used for prokaryotes or other cells thatcontain substantial cell-wall barriers. Infection with Agrobacteriumtumefaciens is used for transformation of certain plant cells, asdescribed by Shaw et al., (1983) Gene, 23: 315 and WO 89/05859 published29 Jun. 1989. For mammalian cells without such cell walls, the calciumphosphate precipitation method described in section 16.30–16.37 ofSambrook et al., supra, is preferred. General aspects of mammalian cellhost system transformations have been described by Axel in U.S. Pat. No.4,399,216 issued Aug. 16, 1983. Transformations into yeast are typicallycarried out according to the method of Van Solingen et al., J. Bact.,130: 946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76: 3829(1979). However, other methods for introducing DNA into cells such as bynuclear injection, electroploration, or by protoplast fusion may also beused.

The host cells used to produce the peptides of invention can be culturedin a variety of media. as described generally in Sambrook et al. A widevariety of transcriptional and translational regulatory sequences may beemployed, depending upon the nature of the host to control theexpression. Transcriptional initiation regulatory signals may beselected which allow for repression or activation, so that expression ofthe gene sequences can be modulated. Of interest are regulatory signals,which are temperature-sensitive so that by varying the temperature,expression can be repressed or initiated, or are subject to chemical(such as metabolite) regulation.

In an intracellular expression system or periplasmic space secretionsystem, the recombinantly expressed peptides of invention can berecovered from the culture cells by disprupting the host cellmembrane/cell wall (eg by osmotic shock or solubilizing the host cellmembrane in detergent). Alternatively, in an extracellular secretionsystem, the recombinant peptide can be recovered from the culturemedium. As a first step, the culture medium or lysate is centrifuged toremove any particulate cell debris. The membrane and soluble proteinfractions are then separated. The Z domain variant peptide can then bepurified from the soluble protein fraction. If the peptide is expressedas a membrane bound species, the membrane bound peptide can be recoveredfrom the membrane fraction by solubilization with detergents. The crudepeptide extract can then be further purified by suitable procedures suchas fractionation on immunoaffinity or ion-exchange columns; ethanolprecipitation; reverse phase HPLC; chromatography on silica or on acation exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammoniumsulfate precipitation; gel filtration using, for example, Sephadex G-75;hydrophobic affinity resins and ligand affinity using IgG ligandimmobilized on a matrix. In vitro transcription/translation systems canalso be employed to produce peptides of the present invention using RNAsderived from the SEQ ID NO: 1 encoding DNA constructs. Cell-freetranslation systems have been used in the biosynthesis of proteins andpeptides, and have become a standard tool in molecular biology forprotein production (In vitro transcription and translation protocols,Methods in Molecular Biology, 37 Edited by M. J. Tymms, 1995, HumanaPress. Inc., Merrick, Translation of exogenous mRNAs in reticulocytelysates, Meth. Enzymol. 101:38 (1983)). Kigawa, T. and Yokohama, S.,“Continuous Cell-Free Protein Synthesis System for CoupledTranscription-Translation” Journal of Biochemistry 110:166–168 (1991),Baranov et al., “Gene expression in a cell-free system on thepreparative scale” (1989) Gene 84:463–466, Kawarasaki et al., “Along-lived batch reaction system of cell-free protein synthesis” (1995)Analytical Biochemistry 226:320–324). Both eukaryotic and prokaryoticcell-free systems can be used for in vitro SEQ ID NO: 1 synthesis. Therabbit reticulocyte (Pelham and Jackson, (1976) Eur. J. Biochem., 67:247–256) and wheat germ lysate (Roberts and Paterson, (1973) Proc. Natl.Acad. Sci., 70: 2330–2334) methods are commonly used eukaryotic in vitrotranslation systems. The E. coli S30 extract method devised by Spirin,A. S. et al., “Continuous Cell-Free Translation System Capable ofProducing Polypeptides in High Yield” (1988) Science 242 (4882):1162–1164, Zubay, (1973) Ann. Rev. Genet., 7:267, and the fractionatedmethod of Gold and Schweiger, (1971) Meth. Enzymol., 20: 537 are widelyused prokaryotic in vitro translation systems.

The expression unit for in vitro synthesis comprises a 5′ untranslatedregion and may additionally comprise a 3′ region. The 5′ untranslatedregion of the expression unit contains a promoter or RNA polymerasebinding sequence, a ribosome binding sequence, and a translationinitiation signal. The 5′ untranslated region (“head”) may also containconvenient restriction sites and a translation enhancer or “Activator”sequence(s). The 3′ region may contain convenient restriction sites anda 3′ tail of a selected sequence. The expression unit may be chemicallysynthesized by protocols well known to those skilled in the art.Alternatively, these elements may be incorporated into one or moreplasmids, amplified in microorganisms, purified by standard procedures,and cut into appropriate fragments with restriction enzymes beforeassembly into the expression unit.

The 5′ untranslated region contains a promoter or RNA polymerase bindingsequence, such as those for the T7, T3, or SP6 RNA polymerase.Positioned downstream of or within the promoter region is a DNAsequence, which codes for a ribosomal binding site. This ribosomebinding site may be specific for prokaryotic ribosomal complexes(including ribosomal RNAs) if a prokaryotic translation procedure isused. However, a preferred embodiment of this invention uses aeukaryotic sequence and an in vitro eukaryotic translation system, suchas the rabbit reticulocyte system (Krawetz et al., 1983 Can. J. Biochem.Cell. Biol. 61:274–286; Merrick, 1983 Meth. Enzymol. 101:38). Aconsensus translation initiation sequence, GCCGCCACCATGG (SEQ ID NO:33), well as other functionally related sequences have been establishedfor vertebrate mRNAs (Kozak, 1987 Nucleic Acids Res, 15:8125–8148). Thissequence or related sequences may be used in the DNA construction todirect protein synthesis in vitro. The ATG triplet in this initiationsequence is the translation initiation codon for methionine; in vitroprotein synthesis is expected to begin at this point.

Between the promoter and translation initiation site, it may bedesirable to place other known sequences, such as translation enhanceror “activator” sequences. For example, Jobling et al. (1988 NucleicAcids Res. 16:4483–4498) showed that the untranslated “leader sequences”from tobacco mosaic virus “stimulated translation significantly” inSP6-generated mRNAs. They also reported that the 36-nucleotide 5′untranslated region of alfalfa mosaic virus RNA 4 increases thetranslational efficiency of barley amylase and human interleukin mRNAs(Jobling and Gehrke, 1987 Nature 325:622–625). Black beetle virus(Nodavirus) RNA 2 (Friesen and Rueckert, J. 1981 Virol. 37:876–886),turnip mosaic virus, and brome mosaic virus coat protein mRNAs (Zagorskiet al., Biochimie 65:127–133, 1983) also translate at high efficiencies.In contrast, certain untranslated leaders severely reduce the expressionof the SP6 RNAs (Jobling et al. (1988 Nucleic Acids Res. 16:4483–4498).

In addition, SEQ ID NO: 1 encoding DNA may be incorporated into the invitro expression unit. Within one embodiment, the expressed polypeptidescontain both carrier polypeptide/peptide and SEG ID NO 1. The carrierpeptide would be useful for quantifying the amount of fusion polypeptideand for purification (given that an antibody against the carrierpolypeptide is available or can be produced). One example is 6His aminoacid sequence (SEQ ID NO: 40); the second is the 11 amino acid SubstanceP, which can be attached as fusion peptides to peptides of theinvention. Anti-6 His (6×His: SEQ ID NO: 40) and anti-Substance Pantibodies are commercially available for detecting and quantifyingfusion proteins. Another example is the eight amino acid marker peptide,“Flag” (Hopp et al., 1988 Bio/Technology 6:1204–1210). A preferable formof the carrier polypeptide is one, which may be cleaved from the novelpolypeptide by simple chemical or enzymatic means.

IV. Derivatives of Peptide

As used herein, the term “amino acid” and any reference to a specificamino acid is meant to include naturally occurring proteogenic aminoacids as well as non-naturally occurring amino acids such as amino acidanalogs. One of skill in the art would know that this definitionincludes, unless otherwise specifically indicated, naturally occurringproteogenic (D) or (L) amino acids, chemically modified amino acids,including amino acid analogs such as penicillamine(3-mercapto-D-valine), naturally occurring non-proteogenic amino acidssuch as norleucine and chemically synthesized compounds that haveproperties known in the art to be characteristic of an amino acid. Asused herein, the term “proteogenic” indicates that the amino acid can beincorporated into a protein in a cell through well-known metabolicpathways.

The choice of including an (L)- or a (D)-amino acid into a peptide ofthe present invention depends, in part, on the desired characteristicsof the peptide. For example, the incorporation of one or more (D)-aminoacids can confer increasing stability on the peptide in vitro or invivo. The incorporation of one or more (D)-amino acids also can increaseor decrease the binding activity of the peptide as determined, forexample, using the binding assays described herein, or other methodswell known in the art. In some cases it is desirable to design a peptidethat retains activity for a short period of time, for example, whendesigning a peptide to administer to a subject. In these cases, theincorporation of one or more (L)-amino acids in the peptide can allowendogenous peptidases in the subject to digest the peptide in vivo,thereby limiting the subject's exposure to an active peptide.

As used herein, the term “amino acid equivalent” refers to compounds,which depart from the structure of the naturally occurring amino acids,but which have substantially the structure of an amino acid, such thatthey can be substituted within a peptide, which retains is biologicalactivity. Thus, for example, amino acid equivalents can include aminoacids having side chain modifications or substitutions, and also includerelated organic acids, amides or the like. The term “amino acid” isintended to include amino acid equivalents. The term “residues” refersboth to amino acids and amino acid equivalents.

As used herein, the term “peptide” is used in its broadest sense torefer to compounds containing amino acid equivalents or other non-aminogroups, while still retaining the desired functional activity of apeptide. Peptide equivalents can differ from conventional peptides bythe replacement of one or more amino acids with related organic acids(such as PABA), amino acids or the like or the substitution ormodification of side chains or functional groups.

It is to be understood that limited modifications can be made to apeptide without destroying its biological function. Thus, modificationof the peptides of the present invention that do not completely destroytheir activity is within the definition of the compound claims as such.Modifications can include, for example, additions, deletions, orsubstitutions of amino acids residues, substitutions with compounds thatmimic amino acid structure or functions, as well as the addition ofchemical moieties such as amino or acetyl groups. The modifications canbe deliberate or accidental, and can be modifications of the compositionor the structure.

As used herein, “binding” refers to the ability of a given peptide tointeract with a receptor such that the interaction between the peptideand the receptor is relatively specific. As used herein, the term“relatively specific” means that the affinity of binding of the receptorand peptide is about 1×10⁻⁵M or less. Therefore, the term “binding” doesnot encompass non-specific binding, such as non-specific adsorption to asurface. Non-specific binding can be readily identified by including theappropriate controls in a binding assay. Methods for determining thebinding affinity are described in the Examples below.

The peptides of the present invention are as well useful when they aremaintained in a constrained secondary conformation. As used herein, theterms “constrained secondary structure,” “stabilized” and“conformationally stabilized” indicate that the peptide bonds comprisingthe peptide are not able to rotate freely but instead are maintained ina relatively fixed structure.

Various methods for constraining the secondary structure of a peptideare well known in the art. For example, peptides such as thosecontaining -Phe-Pro-Gly-Phe-sequence (SEQ ID NO: 37) form -turn, awell-known secondary structure. For example, a peptide can be stabilizedby incorporating it into a sequence that forms a helix such as an alphahelix or a triple helix, according to methods described, for example, byDedhar et al., (1987) J. Cell. Biol. 104:585; by Rhodes et al., (1978)Biochem 17:3442; and by Carbone et al., (1987) J. Immunol 138:1838, eachof which is incorporated herein by reference. Additionally, the peptidescan be incorporated into larger linear, cyclic or branched peptides, solong as their receptor-binding activity is retained. The peptides of thepresent invention may be of any size so long as the VEGFreceptor-binding activity is retained, however, in one embodiment,peptides having twenty or fewer total amino acids are preferred.

A preferred method for constraining the secondary structure of a newlysynthesized linear peptide is to cyclize the peptide using any ofvarious methods well known in the art. For example, a cyclized peptideof the present invention can be prepared by forming a peptide bondbetween non-adjacent amino acid residues as described, for example, bySchiller et al., (1985) Int. J. Pept. Prot. Res. 25:171, which isincorporated herein by reference. Peptides can be synthesized on theMerrifield resin by assembling the linear peptide chain usingN^(α)-Fmoc-amino acids with Boc and tertiary-butyl side chainprotection. Following the release of the peptide from the resin, apeptide bond can be formed between the amino and carboxy termini.

A newly synthesized linear peptide can also be cyclized by the formationof a bond between reactive amino acid side chains. For example, apeptide containing a cysteine-pair can be synthesized and a disulfidebridge can be formed by oxidizing a dilute aqueous solution of thepeptide with K₃[Fe(CN)₆]. Alternatively, a lactam such as an

(

-glutamyl)-lysine bond can be formed between lysine and glutamic acidresidues, a lysinonorleucine bond can be formed between lysine andleucine residues or a dityrosine bond can be formed between two tyrosineresidues. Cyclic peptides can be constructed to contain, for example,four lysine residues, which can form the heterocyclic structure ofdesmosine (see, for example, Devlin, Textbook of Biochemistry 3rd ed.(1992), which is herein incorporated by reference).

Methods for forming these and other bonds are well known in the art andare based on well-known rules of chemical reactivity (Morrison and Boyd,Organic Chemistry, 6th Ed. (Prentice Hall, 1992), which is hereinincorporated by reference).

V. Peptidoimetics

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics” (Fauchere, J. (1986) Adv. Drug Res. 15:29; Veber and Freidinger (1985) TINS p. 392; and Evans et al. (1987) J.Med. Chem 30: 1229, which are incorporated herein by reference) and canbe developed, for example, with the aid of computerized molecularmodeling.

In a preferred embodiment, the present invention provides apharmaceutical composition comprising at least one polypeptide orderivative thereof, wherein the polypeptide or derivative thereof iscapable of specific binding with the high affinity VEGF receptor-1 or aderivative of the VEGF receptor-1 and structural similar receptorscomprises the amino acid sequence of SEQ ID NO: 1 wherein thederivatives of SEQ ID NO: 1 comprises peptidomimetics that are capableof specific binding with the high affinity VEGF receptor-1 or aderivative of the VEGF receptor-1.

Peptide mimetics that are structurally similar to therapeutically usefulpeptides may be used to produce an equivalent therapeutic orprophylactic effect. Generally, peptidomimetics are structurally similarto a paradigm polypeptide (i.e., a polypeptide that has a biochemicalproperty or pharmacological activity), such as SEQ ID NO:1, but have oneor more peptide linkages optionally replaced by a linkage selected fromthe group consisting of: —CH₂—NH—, —CH₂S—, —CH₂—CH.₂—, —CH═CH— (cis andtrans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—, by methods known in the artand further described in the following references: Spatola, A. F. in“Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins,” B.Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A. F.,Vega Data (March 1983), Vol. 1, Issue 3, “Peptide BackboneModifications” (general review); Morley, J. S., Trends Pharm Sci (1980)pp. 463–468 (general review); Hudson, D. et al., (1979) Int J Pept ProtRe 14:177–185 (—CH₂ NH—, —CH₂—CH₂—); Spatola, A. F. et al., (1986) LifeSci 38:1243–1249 (—CH₂—S); Hann, M. M., (1982) J Chem Soc Perkin Trans I307–314 (—CH═CH—, cis and trans); Almquist, R. G. et al., (1980) J MedChem 23: 1392–1398 (—COCH₂—); Jennings-White, C. et al., (1982)Tetrahedron Lett 23:2533 (—COCH₂—); Szelke, M. et al., European Appln.EP 45665 (1982) CA: 97:39405 (1982) (—CH(OH)CH.₂—); Holladay, M. W. etal., (1983) Tetrahedron Lett 24:4401–4404 (—C(OH)CH₂—); and Hruby, V.J., (1982) Life Sci 31:189–199 (—CH₂—S—); each of which is incorporatedherein by reference.

In another embodiment, a particularly preferred non-peptide linkage is—CH.₂NH—. Such peptide mimetics may have significant advantages overpolypeptide embodiments, including, for example: more economicalproduction, greater chemical stability, enhanced pharmacologicalproperties (half-life, absorption, potency, efficacy, etc.), alteredspecificity (e.g., a broad-spectrum of biological activities), reducedantigenicity, and others.

Labeling of peptidomimetics usually involves covalent attachment of oneor more labels, directly or through a spacer (eg., an amide group), tonon-interfering position(s) on the peptidomimetic that are predicted byquantitative structure-activity data and/or molecular modeling. Suchnon-interfering positions generally are positions that do not formdirect contacts with the macromolecules(s) (e.g., are not contact pointsin VEGFR-VEGF complexes) to which the peptidomimetic binds to producethe therapeutic effect. Derivitization (e.g., labelling) ofpeptidomimetics should not substantially interfere with the desiredbiological or pharmacological activity of the peptidomimetic.

A variety of designs for peptide mimetics are possible. For example,cyclic peptides, in which the necessary conformation for binding isstabilized by nonpeptides, are specifically contemplated. U.S. Pat. No.5,192,746 to Lobl, et al., U.S. Pat. No. 5,169,862 to Burke, Jr., et al,U.S. Pat. No. 5,539,085 to Bischoff, et al., U.S. Pat. No. 5,576,423 toAversa, et al., U.S. Pat. No. 5,051,448 to Shashoua, and U.S. Pat. No.5,559,103 to Gaeta, et al., all hereby incorporated by reference,describe multiple methods for creating such compounds. Synthesis ofnonpeptide compounds that mimic peptide sequences is also known in theart. Eldred, et al., (J. Med. Chem. 37:3882 (1994)) describe nonpeptideantagonists that mimic the peptide sequence. Likewise, Ku, et al., (J.Med. Chem. 38:9 (1995)) give further elucidation of the synthesis of aseries of such compounds.

Derivatives of SEQ ID NO:1 can be produced using recombinant nucleicacid molecule techniques. Modifications to a specific peptide may bedeliberate, as through site-directed mutagenesis and amino acidsubstitution during biosynthesis, or may be accidental such as throughmutations in hosts, which produce the peptide. Peptides includingderivatives can be obtained using standard mutagenesis techniques suchas those described in Sambrook et al., Molecular Cloning, Cold SpringHarbor Laboratory Press (1989). For example, Chapter 15 of Sambrookdescribes procedures for site-directed mutagenesis of cloned DNA.

Derivatives of SEQ ID NO: 1 include, but not limited by modificationoccurring during or after translation, for example, by phosphorylation,glycosylation, crosslinking, acylation, proteolytic cleavage, linkage toa therapeutic protein, an antibody molecule, membrane molecule or otherligand (see Ferguson et al., 1988, Annu. Rev. Biochem. 57:285–320).

Specific types of genetically produced derivatives also include, but notlimit by amino acid alterations such as deletions, substitutions,additions, and amino acid modifications. A “deletion” refers to theabsence of one or more amino acid residue(s) in the related peptide. An“addition” refers to the presence of one or more amino acid residue(s)in the related peptide. Additions and deletions to a peptide may be atthe amino terminus, the carboxy terminus, and/or internal, can beproduced by mutation in SEQ ID NO: 1 encoding DNA and/or by peptidepost-translation modification.

Amino acid “modification” refers to the alteration of a naturallyoccurring amino acid to produce a non-naturally occurring amino acid.Analogs of SEQ ID NO: 1 with unnatural amino acids can be created bysite-specific incorporation of unnatural amino acids into polypeptidesduring the biosynthesis, as described in Christopher J. Noren, SpencerJ. Anthony-Cahill, Michael C. Griffith, Peter G. Schultz, 1989 Science,244:182–188.

A “substitution” refers to the replacement of one or more amino acidresidue(s) by another amino acid residue(s) in the peptide. Mutationscan be made in SEQ ID NO: 1 encoding DNA such that a particular codon ischanged to a codon, which codes for a different amino acid. Such amutation is generally made by making the fewest nucleotide changespossible. A substitution mutation of this sort can be made to change anamino acid in the resulting peptide in a non-conservative manner (i.e.,by changing the codon from an amino acid belonging to a grouping ofamino acids having a particular size or characteristic to an amino acidbelonging to another grouping) or in a conservative manner (i.e., bychanging the codon from an amino acid belonging to a grouping of aminoacids having a particular size or characteristic to an amino acidbelonging to the same grouping). Such a conservative change generallyleads to less change in the structure and function of the resultingpeptide. To some extent the following groups contain amino acids whichare interchangeable: the basic amino acids lysine, arginine, andhistidine; the acidic amino acids aspartic and glutamic acids; theneutral polar amino acids serine, threonine, cysteine, glutamine,asparagine and, to a lesser extent, methionine; the nonpolar aliphaticamino acids glycine, alanine, valine, isoleucine, and leucine (however,because of size, glycine and alanine are more closely related andvaline, isoleucine and leucine are more closely related); and thearomatic amino acids phenylalanine, tryptophan, and tyrosine. Inaddition, although classified in different categories, alanine, glycine,and serine seem to be interchangeable to some extent, and cysteineadditionally fits into this group, or may be classified with the polarneutral amino acids. Although proline is a nonpolar neutral amino acid,its replacement represents difficulties because of its effects onconformation. Thus, substitutions by or for proline are not preferred,except when the same or similar conformational results can be obtained.The conformation conferring properties of proline residues may beobtained if one or more of these is substituted by hydroxyproline (Hyp).Derivatives can contain different combinations of alterations includingmore than one alteration and different types of alterations.

The ability of the derivative to retain some activity can be measuredusing techniques described herein and/or using techniques known to thoseskilled in the art for measuring the VEGF receptor-1 binding activity.“Derivatives” of SEQ ID NO: 1 are functional equivalents having similaramino acid sequence and retaining, to some extent, the activities of SEQID NO:1. By “functional equivalent” is meant the derivative has anactivity that can be substituted for the activity of SEQ ID NO:1.Preferred functional equivalents retain the full level of VEGFreceptor-1-binding activity as measured by assays known to these skilledin the art, and/or in the assays described herein. Preferred functionalequivalents have activities that are within 1% to 10,000% of theactivity of SEQ ID NO:1, more preferably between 10% to 1000%, and morepreferably within 50% to 200%. Derivatives have at least 50% sequencesimilarity, preferably 70%, more preferably 90%, and even morepreferably 95% sequence similarity to SEQ ID NO:1. “Sequence similarity”refers to “homology” observed between amino acid sequences in twodifferent polypeptides, irrespective of polypeptide origin.

VI. Biological Agents

A variety of biological agents are suitable for use in the invention.These include, without limitation, proteins, peptides (e.g.,oligopeptides and polypeptides) including cytokines, hormones (such asinsulin), and the like, recombinant soluble receptors, monoclonalantibodies, human growth hormones, tissue plasminogen activators,clotting factors, vaccines, colony stimulating factors, erythropoietins,enzymes, and dismultase. Therefore, in on embodiment, the presentpharmaceutical composition comprising at least one polypeptide orderivative thereof, wherein the polypeptide or derivative thereof iscapable of specific binding with the high affinity VEGF receptor-1 or aderivative of the VEGF receptor-1 and structural similar receptorsfurther comprises a biological agent.

Preferred classes of biological agents (including chemotherapeuticagents) include anti-neoplastic agents, antibacterial agents,antiparasitic agents, anti-fungal agents, CNS agents, immunomodulatorsand cytokines, toxins and neuropeptides. Biological agents for whichtarget cells tend to develop resistance mechanisms are also preferred.Particularly preferred biological agents include anthracyclines such asdoxorubicin, daunorubicin, epirubicin, idarubicin, mithoxanthrone orcarminomycin, vinca alkaloids, mitomycin-type antibiotics,bleomycin-type antibiotics, azole antifungals such as fluconazole,polyene antifungals such as amphotericin B, taxane-relatedantineoplastic agents such as paclitaxel and immunomodulators such astumor necrosis factor alpha (TNF-α), interferons and cytokines.

Preferred biological agents (including chemotherapeutic agents) includewithout limitation additional antifungal agents such as amphotericin-B,flucytosine, ketoconazole, miconazole, itraconazole, griseofulvin,clotrimazole, econazole, terconazole, butoconazole, ciclopirox olamine,haloprogin, toinaftate, naftifine, nystatin, natamycin, undecylenicacid, benzoic acid, salicylic acid, propionic acid and caprylic acid.Such agents further include without limitation antiviral agents such aszidovudine, acyclovir, ganciclovir, vidarabine, idoxuridine,trifluridine, foxcarnet, amantadine, rimantadine and ribavirin. Suchagents further include without limitation antibacterial agents such aspenicillin-related compounds including 9-lactam antibiotics, broadspectrum penicillins and penicillinase-resistant penicillins (such asmethicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin,amoxicillin, ampicillin, ampicillin-sulbactam, azocillin, bacampicillin,carbenicillin, carbenicillin indanyl, cyclacillin, mezlocillin,penicillin G, penicillin V, piperacillin, ticarcillin, imipenem andaztreonam), cephalosporins (cephalosporins include first generationcephalosporins such as cephapirin, cefaxolin, cephalexin, cephradine andcefadroxil; second generation cephalosporins such as cefamandole,cefoxitin, cefaclor, cefuroxime, cefuroxime axetil, cefonicid, cefotetanand ceforanide; third generation cephalosporins such as cefotaxime,ceftizoxime, ceftriaxone, cefoperazone and ceftazidime), tetracyclines(such as demeclocytetracycline, doxycycline, methacycline, minocyclineand oxytetracycline), beta-lactamase inhibitors (such as clavulanicacid), aminoglycosides (such as amikacin, gentamicin C, kanamycin A,neomycin B, netilmicin, streptomycin and tobramycin), chloramphenicol,erythromycin, clindamycin, spectinomycin, vancomycin, bacitracin,isoniazid, rifampin, ethambutol, aminosalicylic acid, pyrazinamide,ethionamide, cycloserine, dapsone, sulfoxone sodium, clofazimine,sulfonamides (such as sulfanilamide, sulfamethoxazole, sulfacetamide,sulfadiazine, and sulfisoxazole), trimethoprim-sulfamethoxazole,quinolones (such as nalidixic acid, cinoxacin, norfloxacin andciprofloxacin), methenamine, nitrofurantoin and phenazopyridine. Suchagents further include agents active against protozoal infections suchas chloroquine, diloxanide furoate, emetine or dehydroemetine,8-hydroxyquinolines, metronidazole, quinacrine, melarsoprol, nifurtimox,pentamidine, sodium stibogluconate and suramin.

A variety of central nervous system agents are suitable for use in thepresent composition. These include neuroleptics such as thephenothiazines (such as compazine, thorazine, promazine, chlorpromazine,acepromazine, aminopromazine, perazine, prochlorperazine,trifluoperazine, and thioproperazine), rauwolfia alkaloids (such asreserpine and deserpine), thioxanthenes (such as chlorprothixene andtiotixene), butyrophenones (such as haloperidol, moperone,trifluoperidol, timiperone, and droperidol), diphenylbutylpiperidines(such as pimozide), and benzamides (such as sulpiride and tiapride);tranquilizers such as glycerol derivatives(such as mephenesin andmethocarbamol), propanediols (such as meprobamate), diphenylmethanederivatives (such as orphenadrine, benzotrapine, and hydroxyzine), andbenzodiazepines(such as chlordiazepoxide and diazpam); hypnotics (suchas zolpdem and butoctamide); 9-blockers (such as propranolol,acebutonol, metoprolol, and pindolol); antidepressants such asdibenzazepines (such as imipramine), dibenzocycloheptenes (such asamitriptyline), and the tetracyclics (such as mianserine); MAOinhibitors (such as phenelzine, iproniazide, and selegeline);psychostimulants such as phenylethylamine derivatives (such asamphetamines, dexamphetamines, fenproporex, phentermine, amfepramone,and pemline) and dimethylaminoethanols (such as clofenciclan,cyprodenate, amninorex, and mazindol); GABA-mimetics (such asprogabide), alkaloids (such as co-dergocrine, dihydroergocristine, andvincamine); cholinergics (such as citicoiine and physosigmine);vasodilators (such as pentoxifyline); and cerebro active agents (such aspyritinol and meclofenoxate); as well as mixtures of several suchagents.

Of particular interest are sedative-hypnotics such as thebenzodiazepines, psycho-pharmacological agents such as thephenothiazines, thioxanthenes, butyrophenones, and dibenzoxazepines, andcentral nervous system stimulants. In one embodiment, the pharmaceuticalcomposition of the present invention further comprises a biologicalagent that can be applied to a wide variety of central nervous systemagents by applying the principles and procedures described herein.

The compositions also can utilize a variety of polypeptides such asantibodies, toxins such as diphtheria toxin, peptide hormones, such ascolony stimulating factor, and tumor necrosis factors, neuropeptides,growth hormone, erythropoietin, and thyroid hormone, lipoproteins suchas α-lipoprotein, proteoglycans such as hyaluronic acid, glycoproteinssuch as gonadotropin hormone, immunomodulators or cytokines such as theinterferons or interleukins, hormone receptors such as the estrogenreceptor. Preferred peptides are those with molecular weight of at leastabout 1,000, more preferably at least about 5,000, most preferably atleast about 10,000. The compositions also can be utilize enzymeinhibiting agents such as reverse transcriptase inhibitors, proteaseinhibitors, angiotensin converting enzymes, 5α-reductase, and the like.Typical of these agents are peptide and nonpeptide structures such asfinasteride, quinapril, ramipril, lisinopril, saquinavir, ritonavir,indinavir, nelfinavir, zidovudine, zalcitabine, allophenylnorstatine,kynostatin, delaviridine, bis-tetrahydrofuran ligands (see, for exampleGhosh et al., J. Med. Chem. 39 (17): 3278–90 1966), and didanosine. Suchagents can be administered alone or in combination therapy; e.g., acombination therapy utilizing saquinavir, zalcitabine, and didanosine orsaquinavir, zalcitabine, and zidovudine. See, for example, Collier etal., Antiviral Res., 1996 Jam. 29 (1): 99.

A variety of human and animal cytokines are suitable for use in thepresent compositions. These include interferons, interleukins, tumornecrosis factors (TNFs) such as TNFα, and a number of other protein andpeptide factors controlling functions of the immune system. It will beappreciated that this extends to mixtures of several such agents, andthe invention is not directed to the underlying specific activity of thecytokines themselves, but rather to the compositions themselves.

VII. Carriers

A variety of carriers can be associated with the ligand including, butnot limiting by synthetic, semi-synthetic and natural compounds such aspolypeptides, lipids, carbohydrates, polyamines, synthetic polymers,that form solutions (unimolecular systems), dispersions (supramolecularsystems), or any particular systems such as nanoparticles, microspheres,matrixes, gels and other.

Therefore, in one embodiment, this invention provides a pharmaceuticalcomposition comprising at least one polypeptide or derivative thereof,wherein said polypeptide or derivative thereof is capable of specificbinding with the high affinity VEGF receptor-1 or a derivative of theVEGF receptor-1 and structural similar receptors further comprises acarrier. The invention further provides a pharmaceutical compositioncomprising one or more Ligands or their complex with a carrier inassociation with a biological agent

The following classes of carriers are given as examples. It isunderstood, however, that a variety of other carriers can be used in thepresent invention.

The polymeric carriers can be nonionic water-soluble, nonionichydrophobic or poorly water soluble, cationic, anionic or polyampholite,such as a polypeptides. It is preferred that the degrees ofpolymerization of these polymer carriers were from about 3 to about500,000 more preferably from about 5 to about 5000, still morepreferably from about 20 to about 500.

Preferred hydrophilic carrier is a nontoxic and non-immunogenic polymerwhich is soluble in water. Such segments include (but not are limitedto) polyethers (e.g., polyethylene oxide), polysaccharides (e.g.,dextran), polyglycerol, homopolymers and copolymers of vinyl monomers(e.g., polyacrylamide, polyacrylic esters (e.g., polyacryloylmorpholine), polymethacrylamide, poly(N-(2-hydroxypropyl)methacrylamide,polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyltriazole, N-oxide ofpolyvinylpyridine, copolymer of vinylpyridine and vinylpyridine N-oxide)polyortho esters, polyaminoacids, polyglycerols (e.g.,poly-2-methyl-2-oxazoline, poly-2-ethyl-2-oxazoline) and copolymers andderivatives thereof.

Preferred nonionic hydrophobic and poorly water soluble segments includepolypropylene oxide, copolymers of polyethylene oxide and polyethyleneoxide, polyalkylene oxide other than polyethylene oxide andpolypropylene oxide, homopolymers and copolymers of styrene (e.g.,polystyrene), homopolymers and copolymers isoprene (e.g., polyisoprene),homopolymers and copolymers butadiene (e.g., polybutadiene),homopolymers and copolymers propylene (e.g., polypropylene),homopolymers and copolymers ethylene (e.g., polyethylene), homopolymersand copolymers of hydrophobic amino acids and derivatives of amino acids(e.g., alanine, valine, isoleucine, leucine, norleucine, phenylalanine,tyrosine, tryptophan, threonine, proline, cistein, methionone, serine,glutamine, aparagine), homopolymers and copolymers of nucleic acid andderivatives thereof.

Preferred polyanionic carrier include those such as polymethacrylic acidand its salts, polyacrylic acid and its salts, copolymers of methacrylicacid and its salts, copolymers of acrylic acid and its salts, heparin,polyphosphate, homopolymers and copolymers of anionic amino acids (e.g.,glutamic acid, aspartic acid), polymalic acid, polylactic acid,polynucleotides, carboxylated dextran, and the like.

Preferred polycationic carrier include polylysine, polyasparagine,homopolymers and copolymers of cationic amino acids (e.g., lysine,arginine, histidine), alkanolamine esters of polymethacrylic acid (e.g.,poly-(dimethylammonioethyl methacrylate), polyamines (e.g., spermine,polyspermine, polyethyleneimine, polypropyleneimine, polybutileneimine,poolypentyleneimine, polyhexyleneimine and copolymers thereof),copolymers of tertiary amines and secondary amines, partially orcompletely quaternized amines, polyvinyl pyridine and the quaternaryammonium salts of the polycation segments. These preferred polycationsegments also include aliphatic, heterocyclic or aromatic ionenes(Rembaum et al., Polymer letters, 1968, 6; 159; Tsutsui, T., InDevelopment in ionic polymers-2, Wilson A. D. and Prosser, H. J. (eds.)Applied Science Publishers, London, N.Y. , vol. 2, pp. 167–187, 1986).

Additionally, dendrimers, for example, polyamidoamines of variousgenerations (Tomalia et al., Angew. Chem., Int. Ed. Engl. 1990, 29, 138)can be also used.

Particularly preferred are copolymers selected from the followingpolymer groups:

-   (a) segmented copolymers having at least one hydrophilic nonionic    polymer and at least one hydrophobic nonionic segment    (“hydrophilic-hydrophobic copolymers”);-   (b) segmented copolymers having at least one cationic segment and at    least one nonionic segment (“cationic copolymers”);-   (c) segmented copolymers having at least one anionic segment and at    least one nonionic segment (“anionic copolymers”);-   (d) segmented copolymers having at least one polypetide segment and    at least one non-peptide segment (“polypeptide copolymers”);-   (e) segmented copolymers having at least one polynucleotide segment    and at least one segment which is not a nucleic acid “polypeptide    copolymers”);

Typical representatives of hydrophilic-hydrophobic copolymers are theblock copolymers of ethylene oxide and propylene oxide having theformulas:

A. Copolymers of Ethylene Oxide and Propylene Oxide.

In one preferred embodiment, the segmented copolymers are the blockcopolymers of ethylene oxide and propylene oxide having the formulas:

in which x, y, z, i and j have values from about 2 to about 800,preferably from about 5 to about 200, more preferably from about 5 toabout 80, and wherein for each R¹, R² pair, one is hydrogen and theother is a methyl group.

Formulas (I) through (III) are oversimplified in that, in practice, theorientation of the isopropylene radicals within the B block will berandom. This random orientation is indicated in formula (IV), which ismore complete. Such poly(oxyethylene)-poly(oxypropylene) compounds havebeen described by Santon, Am. Perfumer Cosmet., 72(4):54–58 (1958);Schmolka, Loc. cit. 82(7):25 (1967); Schick, Non-ionic Surfactants, pp.300–371 (Dekker, N.Y., 1967). A number of such compounds arecommercially available under such generic trade names as “poloxamers”,“pluronics” and “synperonics.” Pluronic polymers within the B-A-Bformula are often referred to as “reversed” pluronics, “Pluronic-R” or“meroxapol.” The “polyoxamine” polymer of formula (XVII) is availablefrom BASF (Wyandotte, Mich.) under the tradename TETRONIC™. The order ofthe polyoxyethylene and polyoxypropylene blocks represented in formula(XVII) can be reversed, creating TETRONIC-R™, also available from BASF.See, Schmolka, J. Am. Oil Soc., 59:110 (1979).Polyoxypropylene-polyoxyethylene block copolymers can also be designedwith hydrophilic blocks comprising a random mix of ethylene oxide andpropylene oxide repeating units. To maintain the hydrophilic characterof the block, ethylene oxide will predominate. Similarly, thehydrophobic block can be a mixture of ethylene oxide and propylene oxiderepeating units. Such block copolymers are available from BASF under thetradename PLURADOT™.

The diamine-linked pluronic of formula (IV) can also be a member of thefamily of diamine-linked polyoxyethylene-polyoxypropylene polymers offormula:

wherein the dashed lines represent symmetrical copies of the polyetherextending off the second nitrogen, R* is an alkylene of 2 to 6 carbons,a cycloalkylene of 5 to 8 carbons or phenylene, for R¹ and R², either(a) both are hydrogen or (b) one is hydrogen and the other is methyl,for R³ and R⁴ either (a) both are hydrogen or (b) one is hydrogen andthe other is methyl, if both of R³ and R⁴ are hydrogen, then one R⁵ andR⁶ is hydrogen and the other is methyl, and if one of R³ and R⁴ ismethyl, then both of R⁵ and R⁶ are hydrogen.

Those of ordinary skill in the art will recognize, in light of thediscussion herein, that even when the practice of the invention isconfined for example, to poly(oxyethylene)-poly(oxypropylene) compounds,the above exemplary formulas are too confining. Thus, the units makingup the first block need not consist solely of ethylene oxide. Similarly,not all of the B-type block need consist solely of propylene oxideunits. Instead, the blocks can incorporate monomers other than thosedefined in formulas (I)–(V), so long as the parameters of the firstembodiment are maintained. Thus, in the simplest of examples, at leastone of the monomers in block A might be substituted with a side chaingroup as previously described.

A variety of other examples of hydrophilic-hyrophobic block copolymershave been synthesized that can be used in the present invention. Thesecopolymers have the general formula A_(n)B_(m), wherein A is thehydrophilic homopolymer or copolymer segment, and B is a hydrophobichomopolymer or copolymer segment. Each of the A and B segments can beeither straight chain or branched. Examples of block copolymers that areparticularly useful in the current invention include, but are notlimited to poly(ethylene oxide)β-poly(isoprene)-β-poly(ethylene oxide)triblock copolymer (Morgan, et al., Biochem. Soc. Trans., 18:1021,1990), poly(ethylene oxide)-β-poly(styrene) block copolymer (Dunn, etal., Pharm. Res., 11:1016, 1994), poly(ethyleneoxide)-β-poly(D,L-lactide) diblock copolymer (Hagan, et al. Langmuir12:2153, 1996), and poly(ethylene oxide)-β-poly((-benzyl L-aspartate)diblock copolymer (Kwon, et al. Langmuir 12:945, 1993).

The hydrophilic homopolymer or copolymer A segments inhydrophilic-hyrophobic block copolymers that can be used in the presentinvention will contain at least three monomeric units, each of whichunit will have the same or different pendant group. Each pendant groupwill contain at least one atom selected from the group consisting ofoxygen and nitrogen. Representative hydrophilic homopolymers orcopolymers include but are not limited to polyethylene oxides,copolymers of ethylene oxide and propylene oxide, polysaccharides,polyacrylamides, polygycerols, polyvinylalcohols, polyvinylpyrrolidones,polyvinylpyridine N-oxides, copolymers of vinylpyridine N-oxide andvinylpyridine, polyoxazolines, and polyacroylmorpholines.

Preferably, the hydrophilic A segment is:

a copolymer of

in which each of m and j has a value of from 3 to 5000.

The hydrophobic B segments useful in this invention can also containfluorocarbon moieties including but not limited to fluoroalkyl segments,and copolymers containing both fluorocarbon and hydrocarbon. One suchexample is the segmented block copolymers having the formula:R¹-L¹-{R²-L²-A}w-L⁴-R⁴-L³-R³  (VI)in which:

-   either, (i) R¹ is a monovalent fluorinated hydrocarbon of 2 to 50    carbon atoms and R² is a divalent hydrocarbon of 2 to 50 carbon    atoms or (ii) R¹ is a monovalent hydrocarbon of 2 to 50 carbon atoms    and R² is a divalent fluorinated hydrocarbon of 2 to 50 carbon    atoms;-   R³ is (i) hydrogen, (ii) a monovalent fluorinated hydrocarbon of 2    to 50 carbon atoms, or (iii) a monovalent hydrocarbon of 2 to 50    carbon atoms;-   R⁴ is (i) a bond if R³ is hydrogen; (ii) a divalent hydrocarbon of 2    to 50 carbon atoms if R³ is a fluorinated hydrocarbon, or (iii) a    divalent fluorinated hydrocarbon of 2 to 50 carbon atoms if R³ is a    hydrocarbon;    -   each of L¹ and L², independently of the other, is a linking        group;    -   L³ and L⁴ taken together with R⁴, is a bond if R³ is hydrogen or        if R³ is other than hydrogen each of L³ and L⁴, taken        independently is a linking group;-   A is a hydrophilic homopolymer or copolymer comprising at least    three monomeric units each having the same or different pendant    group containing at least atom selected from the group consisting of    oxygen and nitrogen; and-   w has a value of from 1 to 100.

The hydrophilic homopolymer or copolymer A will contain at least threemonomeric units, each of which unit will have the same or differentpendant group. Each pendant group will contain at least one atomselected from the group consisting of oxygen and nitrogen.Representative hydrophilic homopolymers or copolymers includepolyethylene oxides, copolymers of ethylene oxide and propylene oxide,polysaccharides, polyacrylamides, polygycerols, polyvinylalcohols,polyvinylpyrrolidones, polyvinylpyridine N-oxides, copolymers ofvinylpyridine N-oxide and vinylpyridine, polyoxazolines, andpolyacroylmorpholines.

B. Cationic Copolymers.

Useful segmented copolymers include a class of copolymers in which atleast one segment is a polycation. One example of these structures is abasis of copolymers comprising a plurality of covalently bound polymersegments wherein the segments have (a) at least one polycation segmentwhich segment is a cationic homopolymer, copolymer, or block copolymercomprising at least three aminoalkylene monomers, the monomers beingselected from the group consisting of at least one of the following:

-   (i) at least one tertiary amino monomer of the formula:

-    and the quaternary salts of the tertiary amino monomer, or (ii) at    least one secondary amino monomer of the formula:

-    and the acid addition and quaternary salts of the secondary amino    monomer, in which:-   R¹ is hydrogen, alkyl of 2 to 8 carbon atoms, an A monomer, or a B    monomer; each of R² and R³, taken independently of the other, is the    same or different straight or branched chain alkanediyl group of the    formula:    —(C_(z)H_(2z))—-    in which z has a value of from 2 to 8; R⁴ is hydrogen satisfying    one bond of the depicted geminally bonded carbon atom; and R⁵ is    hydrogen, alkyl of 2 to 8 carbon atoms, an A monomer, or a B    monomer; R⁶ is hydrogen, alkyl of 2 to 8 carbon atoms, an A monomer,    or a B monomer; R⁷ is a straight or branched chain alkanediyl group    of the formula:    —(C_(z)H_(2z))—-    in which z has a value of from 2 to 8; and R⁸ is hydrogen, alkyl of    2 to 8 carbon atoms, an A monomer, or a B monomer; and-   (b) at least one straight or branched nonionic hydrophilic segment A    having from about 5 to about 1000 monomeric units which is defined    above.

The polycationic segments in the copolymers of the invention can bebranched. For example, polyspermine-based copolymers are branched. Thecationic segment of these copolymers was synthesized by condensation of1,4-dibromobutane and N-(3-aminopropyl)-1,3-propanediamine. Thisreaction yields highly branched polymer products with primary,secondary, and tertiary amines.

An example of branched polycations are products of the condensationreactions between polyamines containing at least 2 nitrogen atoms andalkyl halides containing at least 2 halide atoms (including bromide orchloride). In particular, the branched polycations are produced as aresult of polycondensation. An example of this reaction is the reactionbetween N-(3-aminiopropyl)-1,3-propanediamine and 1,4-dibromobutane,producing polyspermine.

Another example of a branched polycation is polyethyleneiminerepresented by the formula:(NHCH₂CH₂)_(x)[N(CH₂CH₂)CH₂CH₂]_(y)  (VI)

One example of useful polyamine-based copolymers is the polymer offormula:K¹-L¹-[G-L²-F-L³]1-K²,  (VII)in which:

-   F is a polyamine segment comprising a plurality of repeating units    of formula —NH—R⁰, wherein R⁰ is an aliphatic group of 2 to 6 carbon    atoms, which may be substituted;-   G is polyethylene oxide or copolymer ethylene oxide and propylene    oxide a straight or branched nonionic segment defined above;-   K¹ and K² independently of the other, is hydrogen, hydroxy group,    amonogroup, G or F polymer segments;-   and each of L¹, L² and L³, independently of the other, is a linking    group or chemical bond.

The amino groups of polycationic segments can be quaternized to produceammonium salts. Examples include polyspermine and polyamines that aremodified with alkylhalides to produce tertiary and quaternizedpolyamines. Another useful type of cationic segments of well definedchemical structure are ionenes that can be aliphatic, heterocyclic oraromatic (Rembaum et al. Polymer Letters, 1968, 6:159; Tsutsui, T.,Development in ionic polymers-2. Wilson, A. D. and Prosser, H. J.(eds.), Applied Science Publishers, London, N.Y. , vol. 2, pp. 163–187,1986).

C. Anionic Copolymers.

Anionic copolymers contain at least one polyelectrolyte segment thatyields a polyanion in an aqueous environment. This includes both strongpolyacids having high ionization degrees in a broad range of pH, andweak polyacids characterized by pH-dependent ionization degrees. Anionicsegments normally have a plurality of pendant amino groups such ascarobxylic groups, sulfate groups, sulfonate groups, phosphate groups,and the like. Examples of anionic copolymers include but are not limitedto polyoxyethylene-

-polymethacrylic acid (Wang, et al., J. Polym. Sci., Part A: Polym.Chem., 30:2251, 1992), polystyrene-

-polyacrylic acid (Zhong, et al. Macromolecules, 25:7160, 1992),polyacrylic acid grafted withpolyoxyethylene-b-polyoxypropylene-b-polyoxyethylene (Bromberg andLevin, Macromol. Rapid Commun. 17:169 1996).

D. Polypeptide Copolymers.

Polypeptide copolymers have a plurality of covalently bound polymersegments wherein the segments have at least one polypeptide segment andat least one non-peptide polymer segment. Polypeptide segments have aplurality of amino acid units or derivatives thereof.

Examples of useful segmented copolymers containing polypeptides includethe poly(oxyethylene)-poly-L-lysine) diblock copolymer of the followingformula:

wherein i is an integer of from about 2 to about 500, and j is aninteger from about 4 to about 500 ((Lys)j is disclosed as SEQ ID NO:42). A second example is the poly(oxyethylene)-poly-(L-alanine-L-lysine)diblock copolymer of formula:

wherein i is an integer of from about 2 to about 500, and j is aninteger from about 2 to about 500 ((AlaLys)j is disclosed as SEQ ID NO:43).

The use of polypeptide copolymers in the invention allows for bettercontrol of the polypeptide segment lengths by using solid-phase andsolution-phase chemistries. Segmented copolymers based on polypeptideswith well defined chemical structures have been described in theliterature, such as poly(aminoacid)-β-poly(N,N-dietylacrylamide)-β-poly(amino acid) (Bromberg andLevin, Bioconjugate Chem. 9: 40, 1998). Further, the unit compositionand sequence in polypeptides can be varied including hydrophobic,hydrophilic, ionizable, hydrogen and chemical bond forming amino acidsand derivatives thereof to produce broader variability in the basis ofthe segmented copolymers.

E. Polynucleotide Copolymers.

Polynucleotide copolymers have a plurality of covalently bound polymersegments wherein the segments have at least one segment containing atleast three nucleic acid units or the derivatives thereof. Similar topolypeptide copolymers, the polynucleotide copolymers provide for bettercontrol over segment length and sequence by using solid-phase andsolution-phase chemistries. Segmented copolymers based onpolynucleotides with well-defined chemical structure have been describedincluding, polyoxyethylene-β-polynucleotide copolymer andpolycation-β-polynucleotide copolymer (Vinogradov et al., BioconjugateChemistry, 7:3, 1996; U.S. Pat. No. 5,656,611). As with polypeptidecopolymers, polynucleotide copolymers permit variation of the unitcomposition and sequence in polynucleotide segments which isparticularly useful in selecting proper biological agent compositionspursuant to this invention.

VIII. Associating Biological Agents and Carriers with the Ligand

A. Conjugation of the Ligand

In another preferred embodiment the invention provides a Ligand of VEGFreceptor, or derivative of the Ligand, conjugated to a therapeuticagent. Preferred therapeutic agents are described in the abovedescription of invention.

In yet another preferred embodiment the invention provides a Ligand ofVEGF receptor, or derivative of the Ligand, conjugated to a drug carriersystem, such a carrier system being a polymer molecule, a blockcopolymer molecule, or a derivative of said polymer. The carrier systemmay also comprise a protein molecule. Preferred carrier systems aredescribed in the above description of invention.

B. Methods of Chemical Conjugation.

The preparation of the conjugates of the Ligand to the therapeuticagent, or to the carrier system is effected by means of one of the knownorganic chemical methods for chemical ligation. The structural linkbetween the Ligand and the macromolecule, as well as the chemical methodby which they are joined, should be chosen so that the binding abilityof the Ligand and the biological activity of the Ligand, when joined inthe conjugate, are minimally compromised. As will be appreciated bythose skilled in the art, there are a number of suitable chemicalconjugation methods. The selection of the appropriate conjugation methodcan be rationalized by the inspection of the chemical groups present inthe conjugated molecules, as well as evaluation of possible modificationof these molecules to introduce some new chemical groups into them.Numerous chemical groups can subject conjugation reactions. Thefollowing groups are mentioned here as examples: hydroxyl group (—OH),primary and secondary amino group (—NH₂ and —NH—), carboxylic group(—COOH), sulfhydryl group (—SH), aromatic rings, sugar residues,aldehydes (—CHO), alphatic and aromatic halides, and others. Reactivityof these groups is well known in the art (Morrison and Boyd, OrganicChemistry, 6th Ed. (Prentice Hall, 1992), Jerry March, Advanced OrganicChemistry, 4^(th) Ed. (Wiley 1992), which are herein incorporated byreference). A more extensive description of conjugation methods andtechniques can be found in: G. T. Harmanson, Bioconjugate Techniques,Academic Press, Inc. 1995, and in: S. S. Wong, Chemistry of ProteinConjugation and Cross-Linking, CRC Press, Inc. 1991, which are hereinincorporated by reference.

C. Conjugation with Hydroxyl Group

Hydroxyl group —OH is present in peptides and proteins in side chains ofserine, threonine, and tyrosine residues, and in sugar residues insacharides and glycoproteins. Hydroxyl group is also present in manychemical compounds, including therapeutic agents such as paclitaxel, andin polymeric compounds, such as polisacherides and poloxamers. Hydroxylgroups exhibit nucleophilic properties and subject substitutionreaction, for example alkylation (etherification), and acylation(esterification). The following reactive chemicals are preferred toconjugate with hydroxyls: alkyl halides (R—Cl, R—Br), cyanogen bromide(BrCN), acyl anhydrides, acyl halides, aldehydes (—CHO), hydrazides(R—CO—NH—NH₂), and others. Particularly preferred are: acyl anhydrides((R—CO)₂O), and 1,1′-Carbonyldiimidazole (see: Anderson, G. W. and Paul,R., (1958) J. Am. Chem. Soc., 80, 4423, which is herein incorporated byreference).

D. Conjugation with Amino Group

Amino group —NH₂ is present in peptides and proteins at theirN-terminus, if these are not acylated, and in side chains of lysineresidues. Amino group is also present in many chemical compounds,including therapeutic agents such as doxorubicin. Chemical and geneticmethods allow for introduction of amino group into numerous othermolecules, including peptides, proteins, small organic molecules andpolymeric molecules. Amino group reveals nucleophile properties, and itsubjects substitution reaction, for example alkylation, acylation, andcondensation with aldehydes. The following reactive chemicals arepreferred to conjugate with amines: alkyl halides (R—Cl, R—Br, R—I),aryl azides, acyl anhydrides, acyl halides, acyl esters, carboxylatesactivated with carbodiimides, aldehydes (—CHO), and others. Particularlypreferred are: acyl anhydrides ((R—CO)₂O), acyl chlorides (R—CO—Cl),p-nitropheny esters (R—CO—O—C₆H₄—NO₂), N-hydroxysuccinimidyl esters (NHSesters, R—CO—O—N(CO—CH₂)₂), imidoesters (R—C(═NH)—O—CH₃), and carboxylicacids activated with carbodiimides (R—CO—OH+R′—N═C═N—R″).

E. Conjuration with Sulfhydryl Group

Sulfhydryl group —SH is present in peptides and proteins containingcysteine residues. Sulfhydryl group is also present in many chemicalcompounds, and can be introduced into other compounds (see for exampleCarlsson, J., Drevin, H. and Axen, R. (1978) Biochem. J. 173, 723).Sulfhydryl group subjects elecrophilic substitution reaction, forexample alkylation, and oxidation reaction. Preferred are the followingreactive chemicals, useful to conjugate with —SH group: alkyl iodides,unsaturated acyls, and oxidizing agents. Particularly preferred are thefollowing derivatives: iodoacetamides R—CO—CH₂—I, maleimides(R—N(CO—CH)₂), vinylsulfones (R—SO₂—CH═CH₂, Masri M. S. (1988). J.Protein Chem. 7, 49–54, which is herein incorporated by reference),didthiopyridyls (R—S—S-2-pyridyl).

F. Conjugation with Carboxyl Group

Carboxyl group —COOH is present in peptides and proteins at theirC-terminus (if not amidated), and in side chains of aspartic acid andglutamic acid residues. Carboxyl group is also present in many chemicalcompounds, including therapeutic agents such as metotrexate. Chemicaland genetic methods allow for introduction of carboxyl group intonumerous other molecules, including peptides, proteins, small organicmolecules and polymeric molecules. Carboxyl group is able to acylatenucleophilic groups, such as amines and hydroxyls. Carboxyl grouprequires activation prior to conjugation. The preferred methods ofactivation are: reaction with organic or inorganic acid halides (forexample pivaloyl chloride, ethyl chloroformate, thionyl chloride, PCl₅),reaction with carbodiimides (R—CO—OH+R′—N═C═N—R″, for example EDC, DCC),reaction with benzotriazolyl uronium or phosphonium salts (TBTU, BOP,PyBOP).

G. Conjugation with Cross-Linking Reagents

In preferred embodiment the conjugation of the Ligand of VEGF receptorto other molecules, either a therapeutic agent or a drug carriermolecule, is achieved with the support of cross-linking reagent.Particularly preferred are heterobifunctional cross-linking reagents.Variety of cross-linking regents is known to those skilled in the art(see, for example, S. S. Wong, Chemistry of Protein Conjugation andCross-Linking, CRC Press, Inc. 1991. which are herein incorporated byreference).

Heterobifunctional reagents are particularly useful for linking twomolecule, one of them having amino group, and the other havingsulfhydryl group. In a preferred embodiment the Ligand of VEGF has asulfhydryl group, and therefore is available for conjugation withvariety of compounds bearing amino group. The followingheterobifunctonal cross-linking reagents, for example, conjugate aminoto sulfhydryl compounds: GMBS (N-[γ-Maleimidobutyryloxy]succinimideester, Fujiwara, K., et al. (1988); J. Immunol. Meth. 112, 77–83)), SPDP(N-Succinimidyl 3-[2-pyridyldithio]propionate, Carlsson, J., et al.(1978). Biochem. J. 173, 723–737), SIA (N-Succinimidyl iodoacetate,Thorpe, P. E., et al. (1984). Eur. J. Biochem 140, 63–71.), SVSB(N-Succinimidyl-[4-vinylsulfonyl]benzoate).

Particularly preferred heterobifunctional linkers have polyoxyethylenechain between the two reactive groups. Coniugation with such likersyields products having hydrophilic junction between the two conjugatedmolecules, therefore it increases the solubility of the product inaqueous media. The following linkers with polyoxyethylene are mentionedhere as examples: N-Maleimido-polyoxyethylene-succinimide ester(Sharewater Polymers, Cat. No. 2D2Z0F02),vinylsulfone-polyoxyethylene-succinimide ester (Shearewater Polymers,Inc. Al, Cat. No. 2Z5B0F02).

The said biological agents may be used in the invention as biologicallyactive substances. They may as well be used as inactivated chemicalderivatives of biological agents, i.e. prodrugs that are being convertedto the active substances in certain physiological conditions by means ofchemical or enzymatic modification of their structure. For examplepaclitaxel derivatives in which the 2′ or 7-hydroxyl group is convertedinto an ester of a carboxylic acid form a prodrug (Deutsch et al.,“Synthesis of congeners and prodrugs. 3. Water-soluble prodrugs of taxolwith potent antitumor activity,” J. Med. Chem., 32:788–792, 1989.). Forexample doxorubicin derivative in which its amino group is acylated witha carboxylic group of amino acid derivative forms a prodrug (Breistol K,et al. “Superior therapeutic efficacy of N-L-leucyl-doxorubicin versusdoxorubicin in human melanoma xenografts correlates with higher tumourconcentrations of free drug.” Eur J Cancer. 1999 July; 35(7):1143–9.;DeFeo-Jones D, et al. “A peptide-doxorubicin ‘prodrug’ activated byprostate-specific antigen selectively kills prostate tumor cellspositive for prostate-specific antigen in vivo.” Nat. Med 2000 November;6(11):1248–52).

H. Genetical Fusion of Ligand with Therapeutic Polypeptides

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding the Ligand may be ligated toa biological active polypeptide sequence to encode a fusion protein. Thegeneral methods suitable for the construction and expression of Ligandfusions with therapeutic proteins are the same as those described hereinabove for recombinant production of Ligand. Chimeric Ligand-polypeptidesmay be most conveniently constructed by fusing in-frame the DNA sequenceencoding the Ligand of present invention to a cDNA sequence encoding thepolypeptide of interest. However, fusion to genomic fragments oftherapeutic polypeptides can also be used. Alternatively, PCR techniquescan be used to join the two parts of the molecule in-frame with anappropriate vector. Spacer of various length and structure can beinserted between the Ligand sequence and therapeutic protein in order toprovide the fusion protein with additional flexibility and preserve theprotein folding. The fusions of Ligand of the present invention can bepurified by various well-known methods including affinity chromatographyand immobilized metal chelate chromatography (Al-Mashikhi et al., J.Dairy Sci. 71:1756–1763 [1988]). Suitable fusion partners are asdiscussed in Section “Biological agents”.

I. Introduction of Ligand in Virus Proteins

The Ligand of present invention can be introduced into viral particlesin order to change a tropism of virus. Different viruses are capable ofbeing used as vectors for the in vivo transfer and expression of genes.By way of example, retroviruses (RSV, HMS, MMS, and the like), HSVvirus, adeno-associated viruses, adenoviruses, vaccinia virus, and thelike, may be mentioned.

For example, the targeting of adenoviruses can be provided byconstruction of chimeric adenovirus fiber protein, which differs fromthe wild-type coat protein by the introduction of the Ligand amino acidsequence in a conformationally-restrained manner. Such a chimericadenovirus fiber protein will be able to direct entry into cells of avector comprising the chimeric fiber protein that is more efficient thanentry into cells of a vector that is identical except for comprising awild-type adenovirus fiber protein rather than the chimeric adenovirusfiber protein.

Desirably, the Ligand encoding sequence is introduced into the fiberprotein at the level of gene expression. Such the Ligand amino acidsequence either is introduced in place of adenoviral sequences, or inaddition to adenoviral sequences. Regardless of the nature of theintroduction, its integration into an adenoviral fiber protein at thelevel of either DNA or protein, results in the generation of a SEQ IDNO:9 peptide motif in the chimeric fiber protein.

Redirecting viral vectors to VEGF receptor expressing tissues can beachieved by using bispesific conjugates produced by chemical linkage ofanti-virus antibody to the Ligand of present invention, as described,for example for anti-adenovirus antibody (Haisma H J., et al., CancerGene Ther., 2000, Alvarez R D., et al., Clin. Cancer Res., 2000). Toavoid the limitation of chemical conjugations, genetically fusedproteins comprising of anti-fiber knob AB (or cellular adenovirusreceptor, CAR) and the Ligand can be produced (Dmitriev I et al., J.Virol., 2000).

The following examples are intended merely to illustrate the best modenow known for practicing the invention but the invention is not to beconsidered as limited to the details of such examples.

X. Application and Therapeutic Use

In another embodiment of the present invention provides a method oftreating a disease associated with angiogenesis in a patient in need ofsuch therapy comprising administering to the patient an effective amountof a pharmaceutical composition comprising at least one polypeptide orderivative thereof, wherein the polypeptide or derivative thereof iscapable of specific binding with the high affinity VEGF receptor-1 or aderivative of the VEGF receptor-1 and structural similar receptors and apharmaceutical acceptable carrier.

The angiogenesis mediated diseases include, but are not limited to,solid tumors; blood born tumors such as leukemias; tumor metastasis;benign tumors, for example hemangiomas, acoustic neuromas,neurofibromas, trachomas, and yogenic granulomas; rheumatoid arthritis;psoriasis; ocular angiogenic diseases, for example, diabeticretinopathy, retinopathy of prematurity, macular degeneration, cornealgraft rejection, neovascular glaucoma, retrolental fibroplasia,rubeosis; Osler-Webber Syndrome; myocardial angiogenesis; plaqueneovascularization; telangiectasia; hemophiliac joints; angiofibroma;and wound granulation.

The Ligand is useful in the treatment of disease of excessive orabnormal stimulation of endothelial cells. These diseases include, butare not limited to, intestinal adhesions, Crohn's disease,atherosclerosis, scleroderma, and hypertrophic scars, i.e., keloids. TheLigand can be used as a birth control agent by preventingvascularization required for embryo implantation. The Ligand is usefulin the treatment of diseases that have angiogenesis as a pathologicconsequence such as cat scratch disease (Rochele minalia quintosa) andulcers (Helicobacter pylori).

It should be understood that in addition to the ingredients,particularly mentioned above, the formulations of the present inventionmay include other agents conventional in the art having regard to thetype of formulation in question, for example, those suitable for oraladministration may include flavoring agents.

Diseases associated with corneal neovascularization that can be treatedaccording to the present invention include but are not limited to,diabetic retinopathy, retinopathy of prematurity, corneal graftrejection, neovascular glaucoma and retrolental fibroplasia, epidemickeratoconjunctivitis, Vitamin A deficiency, contact lens overwear,atopic keratitis, superior limbic keratitis, pterygium keratitis sicca,sjogrens, acne rosacea, phylectenulosis, syphilis, Mycobacteriainfections, lipid degeneration, chemical burns, bacterial ulcers, fungalulcers, Herpes simplex infections, Herpes zoster infections, protozoaninfections, Kaposi's sarcoma, Mooren's ulcer, Terrien's marginaldegeneration, mariginal keratolysis, trauma, rheumatoid arthritis,systemic lupus, polyarteritis, Wegener's sarcoidosis, scleritis,Stevens-Johnson disease, pemphigoid radial keratotomy, and corneal graphrejection.

Diseases associated with retinal/choroidal neovascularization that canbe treated according to the present invention include, but are notlimited to, diabetic retinopathy, macular degeneration, sickle cellanemia, sarcoid, syphilis, pseudoxanthoma elasticum, Paget's disease,vein occlusion, artery occlusion, carotid obstructive disease, chronicuveitis/vitritis, mycobacterial infections, Lyme's disease, systemiclupus erythematosis, retinopathy of prematurity, Eales' disease,Behcet's disease, infections causing a retinitis or choroiditis,presumed ocular histoplasmosis, Bests disease, myopia, optic pits,Stargardt's disease, pars planitis, chronic retinal detachment,hyperviscosity syndromes, toxoplasmosis, trauma and post-lasercomplications. Other diseases include, but are not limited to, diseasesassociated with rubeosis (neovasculariation of the angle) and diseasescaused by the abnormal proliferation of fibrovascular or fibrous tissueincluding all forms of proliferative vitreoretinopathy, whether or notassociated with diabetes.

Diseases associated with chronic inflammation can be treated by thecompositions and methods of the present invention. Diseases withsymptoms of chronic inflammation include inflammatory bowel diseasessuch as Crohn's disease, ulcerative colitis, psoriasis, sarcoidosis andrheumatoid arthritis. Angiogenesis is a key element that these chronicinflammatory diseases have in common. The chronic inflammation dependson continuous formation of capillary sprouts to maintain an influx ofinflammatory cells. The influx and presence of the inflammatory cellsproduce granulomas and thus, maintains the chronic inflammatory state.Inhibition of angiogenesis by the compositions and methods of thepresent invention inhibit the formation of the granulomas and alleviatethe disease.

The compositions and methods of the present invention can be used totreat patients with inflammatory bowel diseases such as Crohn's diseaseand ulcerative colitis. Both Crohn's disease and ulcerative colitis arecharacterized by chronic inflammation and angiogenesis at various sitesin the gastrointestinal tract. Crohn's disease is characterized bychronic granulomatous inflammation throughout the gastrointestinal tractconsisting of new capillary sprouts surrounded by a cylinder ofinflammatory cells. Inhibition of angiogenesis by the compositions andmethods of the present invention inhibits the formation of the sproutsand prevents the formation of granulomas.

Crohn's disease occurs as a chronic transmural inflammatory disease thatmost commonly affects the distal ileum and colon but may also occur inany part of the gastrointestinal tract from the mouth to the anus andperianal area. Patients with Crohn's disease generally have chronicdiarrhea associated with abdominal pain, fever, anorexia, weight lossand abdominal swelling. Ulcerative colitis is also a chronic,nonspecific, inflammatory and ulcerative disease arising in the colonicmucosa and is characterized by the presence of bloody diarrhea.

The inflammatory bowel diseases also show extraintestinal manifestationssuch as skin lesions. Such lesions are characterized by inflammation andangiogenesis and can occur at many sites other than the gastrointestinaltract. The compositions and methods of the present invention are alsocapable of treating these lesions by preventing the angiogenesis, thusreducing the influx of inflammatory cells and the lesion formation.

Sarcoidosis is another chronic inflammatory disease that ischaracterized as a multisystem granulomatous disorder. The granulomas ofthis disease may form anywhere in the body and thus the symptoms dependon the site of the granulomas and whether the disease active. Thegranulomas are created by the angiogenic capillary sprouts providing aconstant supply of inflammatory cells.

The compositions and methods of the present invention can also treat thechronic inflammatory conditions associated with psoriasis. Psoriasis, askin disease, is another chronic and recurrent disease that ischaracterized by papules and plaques of various sizes. Prevention of theformation of the new blood vessels necessary to maintain thecharacteristic lesions leads to relief from the symptoms.

Another disease, which can be treated according to the present inventionis rheumatoid arthritis. Rheumatoid arthritis is a chronic inflammatorydisease characterized by nonspecific inflammation of the peripheraljoints. It is believed that the blood vessels in the synovial lining ofthe joints undergo angiogenesis. In addition to forming new vascularnetworks, the endothelial cells release factors and reactive oxygenspecies that lead to pannus growth and cartilage destruction. Thefactors involved in angiogenesis may actively contribute to, and helpmaintain, the chronically inflamed state of rheumatoid arthritis. Otherdiseases that can be treated according to the present invention arehemangiomas, Osler-Weber-Rendu disease, or hereditary hemorrhagictelangiectasia, solid or blood borne tumors and acquired immunedeficiency syndrome. In particular, the invention is useful for treatingcancers, including, but not limited to, those cancers exhibiting solidtumors, such as breast, lung, ovarian, testicular, and colon cancersThis invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims. The ligand of the present invention is useful in inhibiting theangiogenic function of target cells both in vitro and in vivo. Theligand of the present invention is particularly useful in inhibiting theangiogenic function of endothelial cells both in vitro and in vivo. Ofparticular interest is the prevention or inhibition of endothelial celldifferentiation into capillary structures. The endothelial cellsamenable to inhibition by the Ligand are present at several sites in amammal and include but are not limited to dermis, epidermis,endometrium, retina, surgical sites, gastrointestinal tract, liver,kidney, reproductive system, skin, bone, muscle, endocrine system,brain, lymphoid system, central nervous system, respiratory system,umbilical cord, breast tissue, urinary tract and the like. The treatmentof the present invention using the ligand is particularly useful inpreventing or inhibiting angiogenesis by endothelial cells at sites ofinflammation and tumorigenesis.

Angiogenesis associated with autoimmune diseases may be treated usingLigand. The autoimmune diseases include but are not limited torheumatoid arthritis, systemic lupus erythematosus, thyroiditis,Goodpasture's syndrome, systemic vasculitis, scleroderma, Sjogren'ssyndrome, sarcoidosis, primary biliary cirrhosis and the like.

Angiogenesis associated with wound repair may also be treated using theligand. Excessive scarring resulting from excess angiogenesis oftenoccurs at sites of skin trauma or surgical sites. Administration of theligand at the site is useful in preventing or inhibiting angiogenesis atthe site to eliminate or lessen the scarring.

The ligand is also useful in methods of inhibiting angiogenesis at asite of tumorigenesis in an immunocompromised mammal. The ligandadministered at such sites prevents or inhibits blood vessel formationat the site thereby inhibiting the development and growth of the tumor.Tumors which may be prevented or inhibited by preventing or inhibitingangiogenesis with the ligand include but are not limited to melanoma,metastases, adenocarcinoma, sarcomas, thymoma, lymphoma, lung tumors,liver tumors, colon tumors, kidney tumors, non-Hodgkins lymphoma,Hodgkins lymphoma, leukemias, uterine tumors, breast tumors, prostatetumors, renal tumors, ovarian tumors, pancreatic tumors, brain tumors,testicular tumors, bone tumors, muscle tumors, tumors of the placenta,gastric tumors and the like.

In the method of treatment, the administration of the ligand, analogs,derivatives or fragments thereof may be for either “prophylactic” or“therapeutic” purpose. When provided prophylactically, the ligand isprovided in advance of any symptom. The prophylactic administration ofthe ligand serves to prevent or inhibit any angiogenesis at a site. Whenprovided therapeutically, the ligand is provided at (or after) the onsetof a symptom or indication of angiogenesis. Thus, the ligand may beprovided either prior to the anticipated angiogenesis at a site or afterthe angiogenesis has begun at a site.

It is proposed that the various compositions of the invention will bebroadly applicable to the treatment or diagnosis of any tumor masshaving a vascular endothelial component. Typical vascularized tumors arethe solid tumors, particularly carcinomas, which require a vascularcomponent for the provision of oxygen and nutrients. Exemplary solidtumors to which the present invention is directed include but are notlimited to carcinomas of the lung, breast, ovary, stomach, pancreas,larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum,cervix, uterus, endometrium, kidney, bladder, prostate, thyroid,squamous cell carcinomas, adenocarcinomas, small cell carcinomas,melanomas, gliomas, neuroblastomas, and the like. The peptides of thepresent invention are useful in the treatment of various neoplastic andnon-neoplastic diseases and disorders. Neoplasms and related conditionsthat are amenable to treatment include carcinomas of the breast, lung,esophagus, gastric anatomy, colon, rectum, liver, ovary, cervix,endometrium, thecomas, arrhenoblastomas, endometrial hyperplasia,endometriosis, fibrosarcomas, choriocarcinoma, head and neck cancer,nasopharyngeal carcinoma, laryngeal carcinoma, hepatoblastoma, Karposi'ssarcoma, melanoma, skin carcinomas, hemangioma, cavernous hemangioma,hemangioblastoma, pancreas carcinoma, retinoblastoma, astrocytoma,glioblastoma, Schwannoma, oligodendroglioma, medulloblastorma,neuroblastomas, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcomas,urinary tract carcinomas, thyroid carcinomas, Wilm's tumor, renal cellcarcinoma, prostate carcinoma, abnormal vascular proliferationassociated with phakomatoses, edema (such as associated with braintumors), and Meigs' syndrome.

Non-neoplastic conditions that are amenable to treatment includerheumatoid arthritis, psoriasis, atherosclerosis, diabetic and otherretinopathies, retrolentral fibroplasia, neovascular glaucoma,age-related macular degeneration, thyroid hyperplasias (includinggrave's disease), corneal and other tissue transplantation, chronicinflammation, lung inflammation, nephrotic syndrome, preclampasia,ascites, pericardial effusion (such as associated with pericarditis) andpleural effusion. The following examples are intended merely toillustrate the best mode now known for practicing the invention but theinvention is not to be considered as limited to the details of suchexamples.

IX. Methods of Use

The ligand compositions of the invention can be administered orally,topically, rectally, vaginally, by pulmonary route by use of an aerosol,or parenterally, i.e. intramuscularly, subcutaneously,intraperitoneallly or intravenously. The ligand composition can beadministered alone, or it can be combined with apharmaceutically-acceptable carrier or excipient according to standardpharmaceutical practice. For the oral mode of administration, the ligandcompositions can be used in the form of tablets, capsules, lozenges,troches, powders, syrups, elixirs, aqueous solutions and suspensions,and the like. In the case of tablets, carriers that can be used includelactose, sodium citrate and salts of phosphoric acid. Variousdisintegrants such as starch, and lubricating agents such as magnesiumstearate, sodium lauryl sulfate and talc, are commonly used in tablets.For oral administration in capsule form, useful diluents are lactose andhigh molecular weight polyethylene glycols. When aqueous suspensions arerequired for oral use, the polynucleotide compositions can be combinedwith emulsifying and suspending agents. If desired, certain sweeteningand/or flavoring agents can be added. For parenteral administration,sterile solutions of the conjugate are usually prepared, and the pH ofthe solutions are suitably adjusted and buffered. For intravenous use,the total concentration of solutes should be controlled to render thepreparation isotonic. For ocular administration, ointments or droppableliquids may be delivered by ocular delivery systems known to the artsuch as applicators or eye droppers. Such compositions can includemucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropylmethylcellulose or poly(vinyl alcohol), preservatives such as sorbicacid, EDTA or benzylchronium chloride, and the usual quantities ofdiluents and/or carriers. For pulmonary administration, diluents and/orcarriers will be selected to be appropriate to allow the formation of anaerosol.

The following examples will serve to further typify the nature of theinvention but should not be construed as a limitation on the scopethereof, which is defined solely by the appended claims.

EXAMPLES Example 1

Solid Phase Peptide Synthesis of the peptides:H-Asn-Gly-Tyr-Glu-Ile-Glu-Trp-Tyr-Ser-Trp-Val-Thr-His-Gly-Met-Tyr-NH₂,(SEQ ID NO: 1)H-Cys-Asn-Gly-Tyr-Glu-Ile-Glu-Trp-Tyr-Ser-Trp-Val-Thr-His-Gly-Met-Tyr-NH₂,(SEQ ID NO: 2)Ac-Cys-Asn-Gly-Tyr-Glu-Ile-Glu-Trp-Tyr-Ser-Trp-Val-Thr-His-Gly-Met-Tyr-NH₂,(SEQ ID NO: 3)Fam-Asn-Gly-Tyr-Glu-Ile-Glu-Trp-Tyr-Ser-Trp-Val-Thr-His-Gly-Met-Tyr-NH₂,(SEQ ID NO: 4)Fam-Glu-Glu-Glu-Asn-Gly-Tyr-Glu-Ile-Glu-Trp-Tyr-Ser-Trp-Val-Thr-His-Gly-Met-Tyr-NH₂,(SEQ ID NO: 5)Fam-Asn-Gly-Tyr-Ile-Glu-Trp-Tyr-Ser-Trp-Val-Thr-His-Gly-Met-Tyr-NH₂.(SEQ ID NO: 6)

For each synthesis the starting material was 0.6 g (0.4 mmol) of RinkAmide resin (4-(2′,4′-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin)substituted at a level of 0.66 mEq per gram of resin (Nova Biochem,Calif.). Each of the L-amino acids, starting with C-terminal tyrosine,was added in sequence in a synthesis cycle consisting of the threesteps: of: piperidine deprotection (step 1), coupling (step 2) andninhydrine test (step 3). If the test showed incomplete coupling, thecoupling step was repeated. The synthesis of peptide SEQ ID NO: 2 wascompleted with additional Fmoc-deprotection (step 1). The synthesis ofpeptide SEQ ID NO: 3 was completed with Fmoc-deprotection (step 1), andacetylation (step 4). The synthesis of peptide SEQ ID NO: 4 wascompleted with Fmoc-deprotection (step 1), and labeling withfluoresceine (step 5). All the completed peptides were subjected totrifluoroacetic acid cleavage (step 6) and then purified (step 7). Allthe operations were performed in a glass reactor with a glass frit fordraining the solvent. The resin was agitated with the solvents and therespective solutions using a shaker rotating the reactor for 180 degree.

1. Fmoc-Deprotection

The Fmoc-protecting group was removed from the starting resin, or fromthe

-amino nitrogen of the amino acid previously attached to the resin, bytreating the resin twice with 20% piperidine in dimethylformamide (DMF)(20 mL) for 3 min, and for 17 min. The resin was then washed six timeswith 10 mL of DMF, each wash taking one minute.

2. Coupling

The appropriate Fmoc-protected amino acid (2.4 mmol dissolved in 7 mLDMF), benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate (PyBOP) (1.25 g dissolved in 3 mL DMF), anddiisopropylethylamine (0.84 mL) was added to the resin, and the mixturewas agitated for 90 minutes. The resin was washed four times with 10 mLDMF. The amino acid derivatives used are represented in Table 1.

TABLE 1 Amino Acid Derivative Tyr Fmoc-Tyr(tBu)-OH Met Fmoc-Met-OH GlyFmoc-Gly-OH His Fmoc-His(Trt)-OH Thr Fmoc-Thr(tBu)-OH Val Fmoc-Val-OHTrp Fmoc-Trp(Boc)-OH Ser Fmoc-Ser(tBu)-OH Glu Fmoc-Glu(OtBu)-OH IleFmoc-Ile-OH Asn Fmoc-Asn(Trt)-OH Cys Fmoc-Cys(Trt)-OH3. Ninhydrine Test

A small sample of the resin (approxymatly 30 beads) was transferred to atest tube. One drop of 1% ninhydrin solution in ethanol, one drop of 80%aqueous phenol, and one drop of 0.001% KCN in pyridine were added to thesample of resin, and the mixture was heated to 120° C. for 5 min. Bluecolor of beads showed incomplete coupling. In this case the couplingstep 2 was repeated. If complete, the synthesis proceeded to the nextcycle.

4. Acetylation of the Peptide SEQ ID NO: 3

After completion of the last cycle with Asn, and Fmoc-deprotection, theresin was agitated with acetic anhydride (0.22 mL) anddiisopropylethylamine (0.84 mL) in 5 mL DMF for 90 minutes at roomtemperature.

5. Labeling of the Peptide SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6with Fluorescein

After completion of the last cycle with Asn, and Fmoc-deprotection, theresin was agitated with 5(and 6-)-carboxyfluorescein (0.9 g dissolved in5 mL DMF), PyBOP (1.25 g dissolved in 3 mL DMF), anddiisopropylethylamine (0.84 mL) for 120 min.

6. Cleavage with Trifluoroacetic Acid

The resin was washed 6 times with DMF, twice with DMF/methanol (1:1v/v), and three times with methanol, and dried in vacuum for 1 hour. Amixture of trifluoroacetic acid (TFA, 9 mL), water (0.5 mL),ethanedithiol (0.25 mL) and triisopropylsilane (0.25 mL) was added tothe dry resin, and was agitated for 2 hours. The liquid was drained, andthe resin was washed with 2 mL TFA. Combined liquids were evaporated ina stream of dry nitrogen. The residue was washed twice with 20 mL ofanhydrous ether, and the peptide was extracted with a 1:1 mixture ofCH₃COOH:H₂O (20 mL), and freeze dried.

7. Purification

The lyophilized powder was dissolved in a mixture CH₃CN:H₂O (1:1, V/V,10 mg of crude peptide in 2 mL) and loaded onto a Vydac C18 preparativecolumn (25×2.25 cm). The loaded column was eluted with a two-componenteluent, 60% of solution A in solution B, at flow rate 5 mL/min. SolutionA was 0.1% TFA in H₂O, and solution B was 0.1% TFA in CH₃CN. Fractionswere identified by electro-spray MS. Fractions exhibiting purity equalto or better than that desired were pooled and lyophilized. The productwas dissolved in CH₃COOH:H₂O (1:1, v/v, 1 mL per 1 mg of product) torender the purified final product as the acetate salt. The products ofthe peptide synthesis are shown in Table 2.

TABLE 2 SEQ ID NO: Yield [mg] m/e (double charge) 1 28 1017.4 2 241069.2 3 20 1091.0 4 7 1195.3 5 2 1391.0 6 10 1132.7

Example 2

Conjugation of peptides with carrier protein The compounds of theinvention are tested for their ability to bind VEGF receptor afterchemical conjugation to the carrier protein.

The carrier protein, for example horseradish peroxidase (ICN, 250 u/mg)was dissolved in phosphate buffer (0.1M Na2HPO4, 0.1M NaCl, 1 mM EDTAand pH 8.5) at final concentration 3 mg/ml.N-succinimidyl-3-(2-pyridylthio)propionate (SPDP, Sigma Chemical) wasdissolved in 133, ul of dimethylformamide (DMFA), in a proportion of0.234 mg SPDP=39 ul DMFA. The solution of SPDP was added to solution ofperoxidase and incubated with stirring at room temperature for 30minutes. After modification, activated protein was purified by gelfiltration. The solution of peroxidase was applied to the Sephadex G-25column (Fisher, 20 ml) and eluted with 50 ml of phosphate buffer. Detectat 280nm with a sensitivity of 50 and lamp intensity of 0.005 Au. Thefractions (1 ml) were collected using a fraction collector (PharmaciaBiotech). The fractions containing modified Peroxidase were selected andcombined (total volume of 5–7 ml). Aliquot of 1 ml was kept for thecontrol. Number of activated groups was evaluated by treatment ofaliquot of activated protein with 1 mg/ml of L-cysteine methyl esterhydrochloride (Aldrich Chemical). Amount of recovered 2-pyridyldisulphide was measured by UV absorbency at 343 nm. Control sample wastreated with cysteine for 15 hours at room temperature, purified by gelfiltration and used as a reference in receptor binding assays, which aredescribed in Example 15. The peptide (SEQ ID NO: 2), 1 mg was dissolvedin 200 μl of phosphate buffer. Activated peroxidase was mixed with thepeptide and incubated with stirring for 24 hours, at room temperature.The reaction was controlled by UV detection at 343 nm (detection of2-pyridyl disulphide). The conjugate was purified by gel filtrationusing Sephadex G-25 column. The conjugate fractions were collected andcombined. The protein concentration was determined using Bradford method(Coomasie blue, Bio-Rad) and conjugation was confirmed by SDS/PAAGelectrophoresis. Peroxidase activity of the conjugate (Conjugate NO 1)per mg of protein was determined by incubation of conjugate aliquotswith ABTS solution (0.22 mg/ml2′2′-azino-bis-93′-ethylbenzthiazoline-6-sulphonic acid) diammoniumsalt, 0.05M citric acid, pH 4.0, 0.05% H₂O₂) for 30 min. at roomtemperature and detection the absorbance at A405.

Example 3

Synthesis of CONJUGATE NO.: 2 (PEG1500-peptide (SEQ ID NO: 3))

The conjugates ID NO 2 was prepared using di-amino derivative ofpolyoxyethylene MW 1500 (PEG-bis-amine MW 1500 from Shearwater Polymers,Inc. Al., Cat. No. PT-017-08). PEG-bis-amine was reacted, with SPDP(N-Succinimidyl 3-[2-pyridyldithio]propionate, from Sigma, Cat. No.P-3415) (step 1). The resulting PEG-bis-SS-pyridyl was purified withHPLC, and it was reacted with excess of peptide SEQ ID NO: 3 (step 2).The progress of the conjugation reaction was monitored by light UVabsorption at 340 nm. Upon completion, the components of the reactionmixture were separated using HPLC.

1. Conjugation of PEG-bis-amine with SPDP.

PEG-bis-amine (15 mg dissolved in 0.2 mL methanol) was mixed with SPDP(5 mg dissolved in 0.1 mL methanol) and diisopropylethylamine (0.027 mLof 10% solution in methanol). The mixture was stirred for 30 minutes,and then it was loaded onto a Vydac C18 preparative column (25×2.25 cm).The column was eluted with a two-component eluent gradient 0.5% perminute, starting from 0% of solution B in solution A, at flow rate 5mL/min. Solution A was 0.1% TFA in H₂O, and solution B was 0.1% TFA inCH₃CN. Fractions were identified by electrospray MS, and by increased UVabsorption at 340 nm after mixing sample with 1% ethanedithiol inmethanol (1:1 v/v). Fractions 50–60 mL after the void volume were pooledtogether and freeze dried, and yield 6.3 mg of PEG-bis-SS-pyridyl.

2. Conjugation of PEG-bis-pyridyl with Peptide SEQ ID NO: 3.

PEG-bis-pyridyl (2 mg dissolved in 1 mL water) was mixed with peptideSEQ ID NO: 3 (6 mg dissolved in 0.1 mL methanol). The mixture wasstirred and its UV absorbance at 340 nm was monitored. After 24 hrs themixture was loaded onto a Vydac C18 preparative column (25×2.25 cm). Thecolumn was eluted with a two-component eluent gradient 0.5% per minute,starting from 0% of solution B in solution A, at flow rate 5 mL/min.Solution A was 0.1% TFA in H₂O, and solution B was 0.1% TFA in CH₃CN.Fractions 110–120 mL after the void volume were pooled together andfreeze dried, and yield 1.1 mg of CONJUGATE NO.: 2 (PEG 1500-peptide(SEQ ID NO: 3)).

Example 4

Synthesis CONJUGATE NO.: 3 (polylysine-PEG1500-peptide (SEQ ID NO: 3))

The conjugates ID NO 3 was prepared using di-amino derivative ofpolyoxyethylene MW 1500 (PEG-bis-amine MW 1500 from Shearwater Polymers,Inc. Al., Cat. No. PT-017-08). PEG-bis-amine was reacted, with SPDP(N-Succinimidyl 3-[2-pyridyldithio]propionate, from Sigma, Cat. No.P-3415), and the resulting PEG-bis-SS-pyridyl was purified with HPLC,(step 1). Polylysine (H-Lys)₈(H-Lys)₄(Lys)₂LysCysNH₂ (SEQ ID NO: 38) wassynthesized according to the protocols described in Example 1 (step 2).Peptide SEQ ID NO: 3 was reacted with excess of PEG-bis-SS-pyridyl,followed by adding of an excess of polylysine to the reaction (step 3).The progress of the conjugation reaction was monitored by light UVabsorption at 340 nm. Upon completion, the components of the reactionmixture were separated using HPLC.

1. Conjugation of PEG-bis-amine with SPDP.

PEG-bis-amine (15 mg dissolved in 0.2 mL methanol) was mixed with SPDP(5 mg dissolved in 0.1 mL methanol) and diisopropylethylamine (0.027 mLof 10% solution in methanol). The mixture was stirred for 30 minutes,and then it was loaded onto a Vydac C18 preparative column (25×2.25 cm).The column was eluted with a two-component eluent gradient 0.5% perminute, starting from 0% of solution B in solution A, at flow rate 5mL/min. Solution A was 0.1% TFA in H₂O, and solution B was 0.1% TFA inCH₃CN. Fractions were identified by electrospray MS, and by increased UVabsorption at 340 nm after mixing sample with 1% ethanedithiol inmethanol (1:1 v/v). Fractions 50–60 mL after the void volume were pooledtogether and freeze dried, and yield 6.3 mg of PEG-bis-SS-pyridyl.

2. Solid Phase Synthesis of Polylysine (H-Lys)₈(H-Lys)₄(Lys)₂LysCysNH₂(SEQ ID NO: 38)

The synthesis of polylysine was performed using 0.5 g (0.1 mmol) ofNovaSyn TGR resin (Nova Biochem, Cat. No. 01-64-0060), usingFmoc-Cys(TRT)-OH for the first cycle of the synthesis, andFmoc-Lys(Fmoc)-OH for next four synthetic cycles. Each synthetic cycleconsisted of steps 1–3 of the Example 1. The reaction was completed withFmoc-deprotection (step 1 of the Example 1), and cleavage (step 6 of theExample 1). The product was purified on HPLC using Vydac C18 preparativecolumn (25×2.25 cm). The column was eluted with a two-component eluentgradient 0.5% per minute, starting from 0% of solution B in solution A,at flow rate 5 mL/min. Solution A was 0.1% TFA in H₂O, and solution Bwas 0.1% TFA in CH₃CN. Fractions were identified by electrospray MS, andby dark blue color developed after mixing sample with 1% ninhydrin inbuthanol (1:1 v/v) at 120° C. for 1 min. The yield of freeze-driedproduct (polylysine) was 21 mg.

3. Conjugation of PEG-bis-pyridyl with Peptide SEQ ID NO: 3.

PEG-bis-pyridyl (3 mg dissolved in 1 mL water) was mixed with peptideSEQ ID NO: 3 (1 mg dissolved in 0.1 mL methanol). UV absorbance of themixture at 340 nm was monitored. After 24 hrs polylysine was added (6 mgdissolved in 0.1 mL water). UV absorbance of the mixture at 340 nm wasfurther monitored. The mixture was stirred for 72 hrs, and then it wasloaded onto a Vydac C18 preparative column (25×2.25 cm). The column waseluted with a two-component eluent gradient 0.5% per minute, startingfrom 0% of solution B in solution A, at flow rate 5 mL/min. Solution Awas 0.1% TFA in H₂O, and solution B was 0.1% TFA in CH₃CN. Fractionswere identified by dark blue color developed after mixing sample with 1%ninhydrin in buthanol (1:1 v/v) at 120° C. for 1 min. Fractions 20–30 mLafter the void volume were pooled together and freeze dried, and yield Img of CONJUGATE NO.: 3 (polylysine-PEG1500-peptide (SEQ ID NO: 3)).

Example 5

Synthesis of CONJUGATE NO.: 4 (PEI-PEG-peptide (SEQ ID NO: 3))

PEI-PEG conjugate was prepared from polyethyleneimine MW ca. 2000 (PEI)and polyethylene glycol MW ca. 8000 (PEG) using carbonyldiimidazol (CDI)as previously described in Kabanov, A. V et al., Bioconjugate Chemistry6, 639–643, 1995. PEI-PEG conjugate (10 mg dissolved in 0.2 mL phosphatebuffer pH 8) was mixed with SPDP (3 mg from Sigma, dissolved in 0.05 mLdimethylformamide,) for 30 minutes. Then the mixture was applied onto aSephadex G-25 column (Fisher, 20 ml) and eluted with 50 ml of phosphatebuffer, and detect with UV absorption at 280 nm. The fraction containingPEI-PEG modified with SS-pyridyl was identified by increased UVabsorption at 340 nm after mixing a sample with 1% ethanedithiol inmethanol (1:1 v/v). This fraction (2 mL) was mixed with peptide SEQ IDNO: 3 (5 mg dissolved in 0.05 mL DMF), and was stirred for 18 hrs. UVabsorbance of the mixture at 340 nm was monitored. Acetic anhydride(0.005 mL) was added to the mixture, and it was stirred for 3 hrs. Thenthe mixture was dialyzed through cellulose membrane (Spectra/Por 1, MWCO6000–8000) against water for 48 hrs. Finally, freeze-drying yielded 4.5mg of the product.

Example 6

Synthesis of CONJUGATE NO.: 5 (paclitaxel-PEG-peptide (SEQ ID NO: 3))

Peptide SEQ ID NO: 2 was conjugated with Fmoc-NH-PEG-COO—N-succinimidyl(MW 3400 from Shearwater Polymers, Inc. Al., Cat. No. 1P2Z0F02),followed by Fmoc group removal with piperidin (step 1). The product waspurified by HPLC. Paclitaxel-2′-succinate was obtained by the methoddescribed by Deutsch H. M. et al. (1989), J. Med. Chem. 32, 788–792.Paclitaxel-2′-succinate was conjugated with NH-PEG-peptide (SEQ ID NO:2) by means of EEDQ (2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, themethod was described by Safavy A. et al. (1999) J. Med. Chem. 42,4919–4924), and produced CONJUGATE NO.: 5 (paclitaxel-PEG-peptide (SEQID NO: 3)) (step 2).

1. Synthesis of amino-PEG-peptide (SEQ ID NO: 2)

Peptide SEQ ID NO: 2 (5 mg dissolved in 2 mL methanol) was mixed withFmoc-NH-PEG-COO—N-succinimidyl (15 mg dissolved in 1 mL methanol) anddiisopropylethylamine (0.01 mL of 10% solution in methanol). The mixturewas stirred for 120 minutes, and then the solvent was removed in vacuum.The residue was dissolved in piperidine (1 mL of 20% solution indimethylformamide), and it was mixed for 20 minutes. The mixture wasthen condensed in vacuum to 0.3 mL, and was diluted with 1 mL 50%aqueous acetonitrile, and it was loaded onto a Vydac C18 preparativecolumn (25×2.25 cm). The column was eluted with a two-component eluentgradient 0.5% per minute, starting from 10% of solution B in solution A,at flow rate 5 mL/min. Solution A was 0.1% TFA in H₂O, and solution Bwas 0.1% TFA in CH₃CN. Fractions were identified by electrospray MS.Fractions 60–80 mL after the void volume were pooled together and freezedried, and yielded 3 mg of amino-PEG-peptide (SEQ ID NO: 2).

2. Synthesis of CONJUGATE NO.: 5 (paclitaxel-PEG-peptide (SEQ ID NO: 3)

Paclitaxel-2′-succinate (2 mg dissolved in 0.2 mL dimethylformamide) andEEDQ (1 mg) were mixed for 30 minutes. Then amino-PEG-peptide (SEQ IDNO: 2) (3 mg in 0.2 mL dimethylformamide) was added, and the mixture wasstirred for 3 hrs. The product was purified on Vydac C18 preparativecolumn (25×2.25 cm) by elution with a two-component eluent gradient 0.5%per minute, starting from 10% of solution B in solution A, at flow rate5 mL/min. Solution A was 0.1% TFA in H₂O, and soution B was 0.1% TFA inCH₃CN. Fractions were identified by electro-spray MS. Fractions 90–100mL after the void volume were pooled together and freeze dried, andyielded 1.2 mg of CONJUGATE NO.: 5 (paclitaxel-PEG-peptide (SEQ ID NO:3)).

Example 7

Synthesis of CONJUGATE NO.: 6 (F127-peptide (SEQ ID NO: 3))

F127-bis-amine, amino derivative of block copolymerpolyoxyethylene-polyoxypropylene-polyoxyethylene (pluronic F127 fromBASF, MW 12600) was prepared using carbonyldiimidazol (CDI) and aqueousammonium (step 1). F127-bis-amine was modified with SPDPheterobifunctional linker to produce F127-bis-SS-pyridyl (step 2). Theconjugate ID NO 6 (F127-peptide (SEQ ID NO: 3)) was then prepared byreaction of F127-bis-SS-pyridyl with peptide SEQ ID NO: 3 (step 3).

1. Synthesis ofpolyoxyethylene-polyoxypropylene-polyoxyethylene-bis-amine(F127-bis-amine)

Pluronic F127 (100 mg dissolved in 3 mL acetonitrile) andcarbonyldiimidazol (26 mg) were mixed for 16 hrs. Then 0.05 mL of conc.aqueous ammonium was added, and the mixing continued for 24 hrs. Thevolatile components were removed in vacuum. The solid residue wasdissolved in 10 mL 1-buthanol and extracted 10 times with 5 mL 1%sulfuric acid in 15% aqueous sodium chloride, 3 times with 1% sodiumbicarbonate in 15% aqueous sodium chloride, and 3 times with 20% aqueoussodium chloride. Finally, the organic phase was evaporated, the residuewas dissolved in water and freeze-dried. The product (70 mg) wasanalyzed by thin layer chromatography (TLC), and showed a single spot onsilica gel plates (Siloca gel 60, Riede-de Haen) developed withchloroform-methanol (7:3 v/v), and visualized with ninhydrine, and withiodine.

2. Synthesis of F127-bis-SS-pyridyl

F127-bis-amine (15 mg dissolved in 0.2 mL methanol) was mixed with SPDP(5 mg dissolved in 0.1 mL methanol) and diisopropylethylamine (0.027 mLof 10% solution in methanol). The mixture was stirred for 30 minutes,and then evaporated. The residue was dissolved in 2 mL of 1-butanol andextracted 3 times with 1 mL 1% sulfuric acid in 15% aqueous sodiumchloride, 6 times with 1% sodium bicarbonate in 15% aqueous sodiumchloride, and 3 times with 20% aqueous sodium chloride. Then, theorganic phase was evaporated, the residue was dissolved in anhydrousethanol, and filtered. The ethanol solution was dried, redissolved inwater and freeze-dried. The product (10 mg) was analyzed by thin layerchromatography (TLC), and showed a single spot on silica gel platesdeveloped with chloroform-methanol (6:4 v/v), visualized with iodine,and not with ninhydrine.

3. Synthesis of Conjugate No.: 6 (F127-peptide (SEQ ID NO: 3))

F127-bis-SS-pyridyl (10 mg dissolved in 1 mL phosphate buffer pH 8) wasmixed with peptide SEQ ID NO: 3 (2 mg dissolved in 0.02 mL DMF), and wasstirred for 18 hrs. UV absorbance of the mixture at 340 nm wasmonitored. Then the mixture was dialysed through cellulose membrane(Spectra/Por 1, MWCO 6000–8000) against water for 48 hrs. Freeze-dryingyield 8 mg of the product.

Example 8

Synthesis of CONJUGATE NO.: 7 (paclitaxel-polyglutamic acid-peptide (SEQID NO: 1))

Paclitaxel-polyglutamic acid (paclitaxel-PG) was prepared frompolyglutamic acid (PG) and paclitaxel using dicyclohexylcarbodiimide(DCC) in dimethylformamide. Paclitaxel-polyglutamnic acid-peptide (SEQID NO: 1) (CONJUGATE. NO.: 7) was prepared from paclitaxel-PG andpeptide (SEQ ID NO: 1) also using dicyclohexylcarbodiimide indimethylformamide.

1. Synthesis of paclitaxel-polyglutainic Acid

Polyglutamic acid sodium salt (MW 50 K, Sigma, 0.5 g) was dissolved inwater. The pH of the aqueous solution was adjusted to 2 using 0.2 M HCl.The precipitate was collected, dialyzed against distilled water, andlyophilized to yield 0.44 g PG. To a solution of polyglutamic acid (100mg, 0.6 mmol of Glu residues) in anhydrous dimethylformamide (2 mL) wasadded paclitaxel (10 mg, 0.011 mmol), dicyclohexylcarbodimide (5 mg,0.025 mmol) and dimethylaminopyridine (0.1 mg). The reaction was allowedto proceed at room temperature for 12 hrs. The reaction was controlledby thin layer chromatography on silica gel plates, usingchlorophormlMeOH (10:1) After 12 hours chromatography showed completeconversion of paclitaxel (Rf=0.5) to polymer conjugate (Rf=0). Thereaction mixture was poured into chloroform. The precipitate wascollected and dried in vacuum, dissolved in distilled water andfreeze-dried to yield 0.1 g of paclitaxel-polyglutamic acid.

2. Synthesis of paclitaxel-polyglutanmc acid-peptide (SEQ ID NO: 1)

Paclitaxel-polyglutamic acid (0.1 g) was dissolved in anhydrousdimethylformamide (2 mL), and dicyclohexylcarbodiimide (3 mg) was added,and stirred gently for 5 minutes. Then peptide (SEQ ID NO: 1) (3 mg) wasadded, and stirred for 12 hours. The reaction mixture was poured intochloroform. The precipitate was collected and dried in vacuum, dissolvedin distilled water and freeze-dried to yield 0.1 g ofpaclitaxel-polyglutamic acid-peptide (SEQ ID NO: 1).

Example 9

Synthesis of CONJUGATE NO.: 8 (paclitaxel-peptide (SEQ ID NO: 1))

Paclitaxel-2′-succinate was obtained by the method described by DeutschH. M. et al. (1989), J. Med. Chem. 32, 788–792. Paclitaxel-2′-succinatewas conjugated with peptide (SEQ ID NO: 1) by means of EEDQ(2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, the method wasdescribed for example by Safavy A. et al. (1999) J. Med. Chem. 42,4919–4924), and produced CONJUGATE ID NO 8 (paclitaxel-peptide(SEQ IDNO: 1)). The product was purified by HPLC.

Paclitaxel-2′-succinate (2mg dissolved in 0.2 mL dimethylformamide) andEEDQ (1 mg) were mixed for 30 minutes. Then peptide (SEQ ID NO: 1) (3 mgin 0.2 mL dimethylformamide) was added, and the mixture was stirred for3 hrs. The product was purified on Vydac C18 preparative column (25×2.25cm) by elution with a two-component eluent gradient 0.5% per minute,starting from 10% of solution B in solution A, at flow rate 5 mL/min.Solution A was 0.1% TFA in H₂O, and solution B was 0.1% TFA in CH₃CN.Fractions were identified by electro-spray MS (m/z 1485.4 doublecharged).

Example 10

Synthesis of CONJUGATE ID. NO.: 9 (peptide (SEQ ID NO:3)-Leucyl-doxorubicin)

1. Modification of Leucyl-doxorubicin

Leucyl-doxorubicin was modified using the heterobifunctional linker SPDPto produce pyridyl-SS-propionylleucyl-doxorubicin (step 1). Theconjugate ID NO 9 (peptide (SEQ ID NO: 3)-Leucyl-doxorubicin) was thenprepared by reaction of pyridyl-SS-propionylleucyl-doxorubicin withpeptide SEQ ID NO: 3 (step 2).

Solution of L-leucyl-doxorubicin (6 mg, 0.01 mmol) in 0.1 mL mathanolwas mixed with SPDP (3 mg) solution in 0.1 mL methanol, anddiispropylethylamine (0.015 mL of 10% solution in methanol). The mixturewas stirred for 20 minutes. The mixture was fractionated using reversephase HPLC with water—acetonitrile gradient (1% per minute, startingfrom 10% acetonitrile). The fractions containing the productpyridyl-SS-propionylleucyl-doxorubicin were identified by MS (m/z 854).

2. Purification

Pyridyl-SS-propionylleucyl-doxorubicin (1 mg dissolved in 0.1 mLdimethylformamide) was mixed with peptide SEQ ID NO: 3 (2 mg dissolvedin 0.02 mL DMF), and was stirred for 18 hrs. The mixture was dilutedwith 2 mL water and freeze dried. The remaining material was purifiedwith using reverse phase HPLC with water—acetonitrile gradient (1% perminute, starting from 10% acetonitrile). The fractions containing theproduct (peptide (SEQ ID NO: 3)-Leucyl-doxorubicin) (CONJUGATE NO.:9)were identified by MS (m/z 1440.5 double charged).

Example 11

Synthesis of the Radioactive Labeled Peptide[³HC]—CO-Cys-Asn-Gly-Tyr-Glu-Ile-Glu-Trp-Tyr-Ser-Trp-Val-Thr-His-Gly-Met-Tyr-NH₂(SEQ ID NO: 3)

The peptide SEQ ID NO: 2 (2 mg) was dissolved in 0.1 mL of 100 mMsolution of sodium bicarbonate. 0.0001 mL of [³H]Acetic anhydride (4–10Ci/mmol, from Amersham Pharmacia Biotech) was added, and the mixture wasstirred for 15 minutes. Then the mixture was acidified with 0.1 mL of200 mM acetic acid, and was applied on 1 mL C-18 solid phase extractioncartridge (Supelco Supelclean LC-18 SPE). The cartridge was washed withwater (2 mL), and the product was eluted with mixture of water andacetonitrile (55:45 v/v), 2 mL. The eluted product showed radioactivityof 0.5 mCi.

Example 12

Peptide SEQ ID NO: 4 Partitioning in Formulation with Polyoxamer F127

The partitioning coefficient P describes the tendency of the peptide tostay formulated within micelles. P=[x]_(m)/[x]_(w), where [x]_(m) is theactual concentration of the substance x inside micelles, [x]_(w) is theactual concentration of the substance x in aqueous phase. Partitioningof the peptide SEQ ID NO: 4 was determined in the formulation consistingof solution of pluronic F127 (BASF) in phosphate buffer saline (PBS, pH7.5) using the fluorescence dependence method (Kabanov A. et al. (1995),Macromolecules 28. 2303–2314). A series of solutions of pluronic F127(concentration ranging from 0.001% to 3% weight/weight) in PBS was usedto dissolve the peptide SEQ ID NO: 4 to obtain the final concentrationof 0.001 mM. The solutions (0.2 mL each) were incubated at 37° C. for 4hrs in 96 well plate (transparent polystyren, flat bottom, fromSarstedt), and the fluorescence of the solution (excitation 485 nM,emission 530 nm) was measured using a plate reader FL600 (BioTek). Thepartitioning constant P was found by fitting the measured fluorescencevalues to the equation:(I _(max) −I ₀)/(I−I ₀)−1=1/(θP)−1/Pwhere:

-   I—fluorescence-   I_(max)—fluorescence at maximum concentration of polymer-   I₀—fluorescence at minimum concentration of polymer-   P—partitioning constant-   ✓=0.01✓([F127]−CMC_(F127)), volume portion of the micellar phase-   ✓—partial specific volume of micelles, approximately ✓=0.8-   [F127]—concentration of the polymer-   CMC_(F127)=0.003% critical micelle concentration of polymer    The same procedure was also applied to fluorescein as a reference    compound.    The results are represented in Table 3.

TABLE 3 Substance Partitioning in F127 Peptide SEQ ID NO: 4 100Fluorescein 40

Example 13

Partitioning of Pyren in Formulation with CONJUGATE NO.: 6 (F127-peptide(SEQ ID NO: 3))

The partitioning coefficient P describes the tendency of pyren, which isan example hydrophobic organic molecule, to stay formulated withinmicelles formed by the polymer mixture containing CONJUGATE ID NO 6. Thepolymer mixture used for the formulation consisted of pluronic F127(BASF) and the CONJUGATE ID NO 6, 90:10 (weight/weight). Assuming thetypical aggregation number for micelles 10–30, the formulation contains2–6 peptide molecules per micelle. P=[x]_(m)/[x]_(w), where [x]_(m) isthe actual concentration of pyren inside micelles, [x]_(w) is the actualconcentration of pyren in aqueous phase. The partitioning was determinedusing the fluorescence dependence method (Kabanov A. et al. (1995),Macromolecules 28. 2303–2314). A series of solutions of the polymermixture (concentration from 0.001% to 3% weight/weight) in PBS pH 7.5was used to dissolve pyren to obtain the final concentration of 0.0005mM. The solutions (0.2 mL each) were incubated at 37° C. for 4 hrs in96-well plate (quarz, Helma), and the fluorescence of the solution(excitation 340 nM, emission 400 nm) was measured using plate readerFL600 (BioTek). The fluorescence dependence on the concentration ofpolymer was analyzed, and the partitioning constant P was found byfitting the measured fluorescence values to the equation:(I _(max) −I ₀)/(I−I ₀)−1=1/(θP)−1/P,where:

-   I—fluorescence-   I_(max)—fluorescence at maximum concentration of polymer-   I₀—fluorescence at minimum concentration of polymer-   P—partitioning constant-   ✓=0.01✓([polymer]−CMC_(polymer)), volume portion of the micellar    phase-   ✓—partial specific volume of micelles, approximately ✓=0.8-   [polymer]—concentration of the polymer-   CMC_(polymer) critical micelle concentration of polymer; CMC of the    mixture of F127 and CONJUGATE ID NO 6 was found to be 0.003%, the    same as for pure F127.    The same procedure was also applied to formulation of pyren in    pluronic F127 as a reference.    The results are represented in Table 4.

TABLE 4 Polymer CMC Partitioning in F127 F127 and 0.003% 1850 CONJUGATE.NO.: 6 (9:1 w/w) F127 0.003% 2000

Example 14

The Ability of Peptides to Bind the VEGF Receptor-1

The compositions of the invention are tested for the binding toimmobilized VEGF receptor-1. To immobilize the VEGF receptor are usedstreptavidin-coated microtiter plates, preblocked with BSA (BoehringerMannheim). Total binding capacity for biotin-labeled AB=1.5 μg/well.Biotin-labeled anti-human FC antibody (ICN) 10 μg/ml in 0.1% BSA/PBS areadded into each well of streptavidin-coated plate (3 wells for eachsample to be tested). The microtiter plates are incubated at 4° C. for 8hours in a humidified container (e.g., a sealed container containingmoistened paper towels). After incubation, the contents of microtiterwells is emptied by inverting plate and flicking the well contents intoa suitable container, tapping the inverted plate on a paper towel toremove any liquid remaining in the wells. Then, microtiter plates arewashed for 4 times with 300 μl of 0.1% BSA/PBS. Recombinant humanFlt-1/Fc chimera (R&D Systems) is used as a source of VEGF receptor-1.The cDNA sequence for the endogenously encoded soluble human Flt-1,containing six IgG-like extracellular domains of Flt-1 receptor(Shibuya, M. et. al., 1990, Oncogene 5: 519–524; Kendall, R. L. and K.A. Thomas 1993, Proc. Natl. Acad. Sci. USA 90: 10705–10709) was fused tothe Fc region of human IgG₁. The recombinant soluble Flt-1/Fc chimerabinds VEGF and PlGF with high affinity according to suppliercertificate. The Flt-1/Fc chimera (1 μg/100 μl in 0.1% BSA/PBS) is addedinto each well. Plates are incubated at 4° C. overnight in sealedcontainer to let receptor to immobilize. Unbound receptor is washed awaywith 2 ml of 0.1% BSA/PBS. Then each well is filled with 250 μl ofBlocking Buffer (2% nonfat dry milk in PBS) and blocked for 2–3 hours atroom temperature. As a negative control, 3 wells with immobilized antiFc-antibody are blocked with 2% nonfat milk in PBS. Fluorescentlylabeled peptide (SEQ ID NO: 4) is resolved in 0.1% BSA/PBS buffer, pH8.5 and added at various concentrations (0 μM, 0.1 μM, 1 μM, 10 μM, 50μM and 100 μM) into wells with immobilized Fit-1 receptor. To evaluatethe receptor binding of chemically modified peptides (SEQ ID NO: 2, SEQID NO: 3), equal concentration of fluorescently labeled peptide andanalyzed peptide are mixed and added into wells. To evaluate the abilityto bind Flt-1 receptor for peptide SEQ ID NO:5 the fluorescein labeledpeptide SEQ ID NO: 5 was added to immobilized receptor at concentration50 μM. The specificity of ligand:receptor interaction is detected byinhibition of fluorescent labeled peptide binding to the receptor in thepresence of free peptide (SEQ ID NO: 1). After 2 hours incubation,microtiter plates with bound peptide are washed 10 times with 0.1%BSA/PBS buffer, pH 8.5 and the fluorescence is measured using MicroplateFluorescence Reader FL600 (BioTek) (λ_(ex)=485 nm; λ_(em)=530 nm).Results are presented in. The binding of the peptide to the immobilizedVEGF receptor-1 is concentration-dependent in comparison to controlpeptide. The analysis demonstrates that SEQ ID NO: 4 has high affinitybinding for VEGF receptor-1 with a K_(d) value of about 10 μM. 64% ofthe binding of fluorescent labeled peptide to immobilized VEGFreceptor-1 was displaced by chemically modified peptide (SEQ ID NO: 3).

TABLE 5 Analyzed compound Concentration in μM Fluorescence SEQ ID NO: 40 0 0.1 512 1 932 10 3375.5 50 4675.5 100 6706 SEQ ID NO: 4 + 50 3707.7SEQ ID NO: 1 50 SEQ ID NO: 4 + 50 1683.2 SEQ ID NO: 3 50 Control peptide0 0 0.1 7 1 20 10 493 100 1105 SEQ ID NO: 5 50 5124.5

Example 15

The Ability of Conjugate NO.: 1 to interact with VEGF Receptor-1

The ability of Conjugate ID NO:1 to interact with VEGF receptor-1 wasanalyzed in receptor binding assay. Human Flt-1/Fc chimera wasimmobilized in microtiter plates as described in Example 14. ConjuguateID NO 1 at various concentrations (0.3 nM; 0.9 nM; 2.8 nM; 8.4 nM; 25.25nM; 75.75 nM; 227.3 nM; 681.8 nM) in 0.1% BSA/PBS, pH 7.5 was added intowells and incubated for 2 hours at room temperature. Unbound conjugatewas washed with 0.1% BSA/PBS, pH 7.5 and the bound peroxidase activitywas detected using ABTS/H₂O₂ reagent, as described above. Peroxidaseconjugated with cysteine was used as control of specific binding. Thedata represent means from triplicate wells (Table 6). Peptide (SEQ IDNO: 4)-peroxidase conjugate demonstrated concentration-dependentspecific binding to the VEGF receptor-1. The half-maximum bindingconcentration for conjugate NO.:1 (0.1 μM) was 50-fold higher than forpeptide.

TABLE 6 Receptor-bound Peroxidase Analyzed compound Concentration in nMactivity, A₄₀₅ Conjugate NO.: 1 0.312 0.019 0.935 0.017 2.8 0.031 8.420.014 25.25 0.092 75.75 0.377 227.3 0.941 681.8 1.162 Cys-Peroxidase227.3 0.022 conjugate (Control) 681.8 0.020

Example 16

Construction of the Phase Expressing SEQ ID NO: 1

The SEQ ID NO: 1 is expressed as fusion protein with minor coat proteinIII of E.coli bacteriophage M13. The phage vector fUSE5, containing SfiI restriction sites in N-end of pIII was kindly provided by Dr. GeorgeSmith, University of Missouri, Columbia Mo. The DNA of fUSE5 (100 μg)vector was digested with Sfi I restrictase (Boehringer MannheimBiochemica) at supplier recommended conditions, purified by phenol:chloroform extraction and isolated from 14-bp stuffer fragment byprecipitation with isopropanol for 20 min on ice. The linearized vectorcontained two non-complementary Sfi I ends, can not be self ligated andallows oriented ligation of oligonucleotides with the appropriatecohesive ends. The oligonucleotide inserts coding SEQ ID NO: 1 andcontrol peptides were synthesized by automatic solid phaseoligonucleotide synthesis and purified by reverse phase chromatography.The sequences of oligonucleotides are represented in Sequence Listing.The 5′- and 3′-ends of oligonucleotide SEQ ID NO: 9; 10 and SEQ ID NO:11 were annealed to two “half-site” fragments SEQ ID NO: 12 and SEQ IDNO: 13 to form cohesive termini complementary to Sfi I sites 1 and 2 inthe vector.

Oligonucleotides were phosphorylated with T4 kinase, and annealed in 20mM Tris-HCl, pH 7.5, 2 mM MgCl₂, 50 mM NaCl, by mixing 1.5 μg SEQ ID NO:12, 1.2 μg SEQ ID NO: 13, and 0.5 μg SEQ ID NO: 9 (or other), heating to65.degree. C. for 5 minutes and allowing the mixture to cool slowly toroom temperature. This represented an approximate molar ratio of5:100:100 (SEQ ID NO:9-SEQ ID NO:10: SEQ ID NO:11). The annealedoligonucleotide insert (200 ng) is then ligated to 20 μg of Sfi-I-cutfUSE5 RF DNA (molar ratio 1:5) to produce a double-stranded circularmolecule with a small, single stranded gap. The annealed DNA was ligatedin 20 mM Tris-HCl, pH 7.5, 5 mM MgCl.sub.2, 2 mM DTT, 1 mM ATP, by theaddition of 20 units of T4 DNA ligase and incubated overnight at15.degree. C. The ligated DNA was ethanol precipitated in the presenceof 0.3M sodium acetate, resuspended in water and electro-transformedinto competent E. coli MC1061 cells using a Gene Pulser electroporationapparatus (Bio Rad) at 1.8 kV/cm, 200Ω, 25 mF. After electroporation,E.coli cells were allowed to reparate at 37° C. for 1 hour in 2 ml ofSOC medium (2% Bacto tryptone, 0.5% Bacto yeast extract, 10 mM NaCl, 2.5mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mM glucose, 0.2 mg/ml tetracycline)and plated on Petri dishes with Luria-Bertani (LB) agar, containing 100mg/ml kanamycin and 40 mg/ml tetracycline. Plates were incubatedovernight at 37° C. Phage from single colonies were amplified in LBmedium and purified using double precipitation with polyethylene glycol,as described in Phage display of peptides and proteins, Ed. by B. Kay etal., Academic Press. San Diego, 1996. The structure of recombinant phageclones was confirmed by dideoxy DNA sequencing.

List of Oligonucleotide Sequences

-   (1) SEQ ID NO: 9 (ONV5.2)-   (A) LENGTH: 69 nucleotides-   (B) TYPE: nucleotide

GG GCC GGT AAC GGG TAC GAG ATC GAG TGG TAC TCG TGG GTC ACG CACGGG ATG TAC GGT GGC GCT TCT G     Gln Gly Tyr Glu Ile Glu Trp Tyr SerTrp Val Thr His Gly Met Tyr (SEQ ID NO:34)

-   (2) SEQ ID NO: 10 (ONV5.10)-   (A) LENGTH: 69 nucleotides-   (B) TYPE: nucleotide

GG GCC GGT CCG GAG CCC GAG GTC CGG TTG AGT CCG CCG GGT CAT ATC CAG TCGCTC GGT GGC GCT TCT G      Pro Glu Pro Glu Val Arg Leu Ser Pro Pro GlyHis Ile Gln Ser Leu (SEQ ID NO:35)

-   (3) SEQ ID NO: 11. (ONV40)-   (A) LENGTH: 69 nucleotides-   (B) TYPE: nucleotide

GG GCC GGT TTT GTG GGG GGG TGG TTG GTT CCG GAG GAC GAG CGG CTCTAC CCG GAG GGT GGC GCT TCT G        Phe Val Gly Gly Trp Leu Val Pro GluAsp Glu Arg Leu Tyr Pro Glu (SEQ ID NO:36)

-   (4) SEQ ID NO: 12 (ON 10)-   (A) LENGTH: 10 nucleotides-   (B) TYPE: nucleotide

AAGCGCCACC

-   (5) SEQ ID NO: 13 (ON 11)-   (A) LENGTH: 11 nucleotides-   (B) TYPE: nucleotide

ACCGGCCCCGT

Example 17

The Binding of SEQ ID NO: 1-Expressing Phase to VEGF Receptor-1

Binding assays with the SEQ ID NO: 1-expressing phage (V5.2) generatedin Example 16 above were performed essentially as described by B. Kayet. al in Phage display of peptides and proteins, Academic Press, SanDiego, 1996. Specifically, binding assays were performed inninety-six-well breakaway streptavidin-coated microliter plates,preblocked with BSA (Boehringer Mannheim). Total binding capacity forbiotin-labeled AB=1.5 μg/well. Immunoabsorbent assay plates were coatedovernight at 4.degree. C. with 10 .mu.g/mI affinity-purified,biotin-labeled goat anti-human Fc IgG (ICN). Human Flt-1/Fc chimera (1μg/200 μl in 0.1% BSA/PBS) was added into each well, and plate wasincubated at 4° C. overnight in sealed container. Plates were blockedfor 2 hours with 2% non-fat milk in PBS (Blocking Buffer). Phagesuspensions diluted in Blocking Buffer to 10⁹ virions/200 μl wereincubated for 30 min at room temperature. This step was necessary toblock any non-specific protein-protein interactions that may occurbetween phages and surrounding proteins. After removal of the blockingbuffer from microtiter plate, 100 μl of a phage suspension with orwithout competing compound was added to each well. The phage was allowedto bind immobilized receptor overnight at 4.degree. Three well for eachphage clone were coated with anti-Fc antibody and used as control.Non-bound phage was removed by washing wells 10 times with 250 μl ofPBS/0.05% Tween 20. Horseradish peroxidase conjugated mouse anti-M13monoclonal antibody (Pharmacia/Biotech) was used to analyzed thebinding. Peroxidase activity was detected by production a green color inthe presence of ABTS/H2O2 reagent and quantified by reading theabsorbance at 405 nm in plate reader FL600 (BioTek). These resultsdemonstrated that SEQ ID NO: 1 expressed as a fusion protein on thesurface of phage particle retained the specific binding to the VEGFreceptor-1 (Table 8.

TABLE 8 N PHAGE CLONE ABSORBANCE, A 405 1 V5.2 1.67 ± 0.09 2 V5.10 0.039± 0.013 3 V40 0.025 ± 0.014

Example 18

The Selectivity of SEQ ID NO:1-Expressing Phase to Human and Murine VEGFReceptor-1

The ability of SEQ ID NO: 1 selectively recognize the VEGF receptor-1was examined using phage ELISA assay. Microtiter ELISA plates (Maxisorb,Nunc) were coated with human and rodent endothelial receptors (1μg/well) in 100 μl of 50 mM Na₂ CO₃, pH 9.6 overnight at 4° C. Plateswere blocked for 2 hours with 2% non-fat milk in PBS to preventnonspecific binding. Then the SEQ ID NO: 1-expressing phage (1×10⁹cfu/wel) was added to each well and allowed to bind immobilizedreceptors for 3 hours at 4.degree C. Non-bound phage was removed byrepeated washings with PBS/0.05% Tween-20 and the amount of remain phagewas determined with anti-M13 antibody as described above. The results ofthe binding assays employing the VEGF receptor-1 Flt-1 and the fourother proteins are presented in Table 5. The SEQ ID NO: 1-expressingphage was highly selective to VEGF receptor-1, did not react with otherhuman endothelial receptors, including VEGF receptor-2, which has 40%sequence homology with VEGF receptor-1. The cross-interaction of SEQ IDNO: 1 phage with murine VEGF receptor-1 was significant and selective.

TABLE 9 No Immobilized protein Absorbance, A 405 1 Human VEGFreceptor-1/Flt-1 0.614 ± 0.005 2 Human VEGF receptor-2/KDR 0.085 ± 0.0163 Human sICAM-1  0.01 ± 0.029 4 Anti-humanFc IgG 0.051 ± 0.014 5 MurineVEGF receptor-1/Flt-1 0.398 ± 0.006 6 Murine VEGF receptor-2/Flk 0.0097± 0.002  7 Rat Neuropilin-1 0.278 ± 0.015

Example 19

The Binding of SEQ ID NO: 3-Polymer Conjugates to VEGF Receptor-1

The binding of SEQ ID NO: 3 conjugates with polymers (CON. NO.:2; CON.NO.:3; CON. NO.: 4), described in examples 3, 4 and 5, was analyzedusing competitive receptor binding assay. The microtiter plates, coatedwith hrVEGF receptor-1 (1 μg/well) were prepared as described in Example14. The SEQ ID NO: 1-expressing phage was used as competing agent. Phagesuspension (10⁹ cfu/ml) in 0.1% BSA/PBS was prepared and mixed withconjugates at various concentrations. The SEQ ID NO: 1-expressing phagealong (Positive control) and mixture of phage with conjugates (200μl/well) were added into microtiter ptates with immobilized receptor,incubated at room temperature for 4 hours. Non-bound phage was removedby washings wells 10 times with 250 μl of 0.1% BSA/PBS. The binding wasdetected using Recombinant Phage Detection Module (Pharmacia Biotech).Result of analysis are presented in Table 10.

TABLE 10 No Analyzed compound Concentration, μM % of inhibition 1Conjugate NO.: 2 50 28.76 2 Conjugate NO.: 3 50 60.91 3 Conjugate NO.: 40.1 7.32

Example 20

Inhibition of VEGF Binding to VEGF Receptor-1 by SEQ ID NO:1 Peptide andSEQ ID NO: 1-Expressing Phage

The ability of SEQ ID NO:1 peptide and SEQ ID NO: 1-expressing phage toinhibit the vascular endothelial factor binding to the VEGF receptor-1was analyzed using competitive receptor binding assay. Human VEGFreceptor-1 coated plates were prepared as described in Example 11.Binding cocktails consisted of 5×10⁹ cfu/ml SEQ ID NO:1-expressing phageor 30 μM of fluorescein labeled peptide (SEQ ID NO: 3); a variousconcentrations of human recombinant VEGF (R&D Systems), all within 0.1%BSA/PBS (Buffer B) for a final volume of 100 μl were assembled and addedto wells with immobilized VEGF receptor-1 or anti-Fc antibody (Control).The cocktails were allowed to equilibrate for 4 hours at roomtemperature. After incubation, micrititer plates were washed with bufferB for 10 times. Fluorescein-labeled peptide (SEQ ID NO: 3) interactionwith the receptor was detected by fluorescence reader as described inExample 11. SEQ ID NO:1-expressing phage binding to immobilized VEGFreceptor-1 was analyzed by ELISA as described in Example 14. The bindingwas calculated in % to Positive control (VEGF receptor-1-bound phage orpeptide in absence of competitor (VEGF)). The results are represented asaverage of triplicate measurement ±SD. As shown in Table 11, both SEQ IDNO: 3 peptide and SEQ ID NO: 1-expressing phage competed with VEGF forthe high-affinity receptor binding. The half-maximum inhibition of SEQID NO: 3 peptide binding was detected in the presence of 5 nM VEGF.

TABLE 11 VEGFR1 VEGF concentration, nM VEGFR1 bound phage, % boundpeptide, % 0 100 100 0.01 99.87 ± 0.69  96.21 ± 5.16  0.1 97.99 ± 3.77 79.34 ± 5.33  1 84.78 ± 0.63  61.44 ± 8.42  10 68.4 ± 2.55 40.8 ± 42  100 25.2 ± 1.24 15.41 ± 11.4 

Example 21 SEQ ID NO:1-Expressing Phase Distribution in Tumor-BearingMice

Murine B16BL6 melanoma cells were cultured in D-MEM supplemented with10% of FBS at 37° C. in humidified atmosphere with 5% CO2. The cellswith a volume of 200 μl of PBS were implanted s.c. in female C57BL/6mice. Two weeks after tumor implantation, the animals were injected i.v.with 1×10¹⁰ virions of SEQ ID NO 1-expressing phage or control phage(random peptide phage library). The phage was allowed to circulate for24 h and then animals were perfused through the heart with 10 ml ofD-MEM medium. The tumor and organs from 3 mice per group were collected,weighted and homogenized. The phage titer in different organs wasdetermined as described (Pasqualini R. et. al., Nature 380:364–366,1996). The titer of recovered phage (cell transducting units per gram oftissue) from tumor, liver and brain is presented in Table 6. The100-fold higher accumulation of the SEQ ID NO 1-expressing phage in thetumor compared to control phage was detected. The maximum accumulationof phage in mice injected with the ligand-expressing phage wasdetermined in tumor (ratio tumor:liver=80.7). Control phage was moreequally distributed in tumor and organs (ratio tumor:liver=4.29)

TABLE 12 SEQ ID NO: 1- expressing phage Control phage Organ CFU × 10⁶/gof tissue CFU × 10⁶/g of tissue Tumor 26000 ± 926  368 ± 112 Liver  323± 14.9 85.8 ± 4.86 Brain 22.1 ± 9.6  7.68 ± 1.55

Example 22

Ability of Peptides to Inhibit the Endothelial Cell Proliferation

Endothelial cells, for example, human umbilical vein endothelial cells(HUVEC) which can be prepared or obtained commercially (Clonetics, SanDiego, Calif.) are plated onto 96-well plates (Costar) at 10⁴ cells perwell in 200 μl of EGM-2 medium (Clonetics) supplemented with 0.5%heat-inactivated fetal bovine serum. Cells are allowed to grow for 24hours at 37° C. under 5% CO₂. Recombinant human VEGF is added to thewells (at 10 ng/ml final concentration) along or with the test compound(SEQ ID NO: 4) at various concentrations: 0 uM; 0.5 uM; 1 uM; 5 uM; 25uM; 50 uM. Non-relevant peptide YAFGYPS (SEQ ID NO: 39) at the sameconcentration is used as control. After 48 hours of incubation at 37° C.under 5% CO₂, 1 μgCi of [methyl-³H]-thymidine (20 Ci/mmol; ICN) is addedper well and plates are incubated for an additional 24 hours. The cellsare then placed on ice, washed twice with EGM-2 medium containing 10%FBS and fixed for 10 minutes by adding 200 μl of ice-cold 10%trichloroacetic acid per well. After washing with ice-cold water, cellsare lysed and DNA is solubilized in 50 μl of 2% SDS. [³H]-Thymidineincorporation is determined by scintillation counting. Results arepresented in Table 8 and expressed as the average of 3 different wellsfor each concentration of compound.

The results show the test compound inhibits endothelial cellproliferation with an IC.sub.50 of 5 μM. At higher concentration of thetest compound the VEGF induced HUVEC proliferation was reduced by 70%.

TABLE 13 VEGF-induced HUVEC Analyzed compound Concentration in μMproliferation in % to control SEQ ID NO: 4 0 100 0.5 75 1 51 5 61 25 3250 27 Control peptide 0 100 0.5 96.7 1 123.5 5 93.8 25 107.4 50 137

Example 23

Inhibition of Tumor-Induced Angiogenesis In Vivo by Peptides

For tumor-induced angiogenesis, the dorsal air sac-chamber was carriedout in female C57BL/6 mice as described else (Itoh, T et al., CancerRes., 58: 1048–1051, 1998). Murine B16BL6 cell, a VEGF-producingmelanoma, were washed three times and suspended in HBSS at aconcentration of 5×10⁶ cells/ml. A Millipore (Millipore Co., Redford,Mass.) was filed with 0.2 ml of either a cell suspension of HBSS andthen implanted s.c. into the dorsal side of mice (day 0). Control micewere implanted with Millipore chambre along. The animals (5 mice pergroup) were treated s.c. with 20 mg/kg of the modified peptide (SEQ IDNO: 4) or the vehicle into 1 cm around the air sac. Treatments wereperformed daily from days 0 to 5.

To evaluate the antiangiogenic activity of the compounds, 7 days aftertumor implantation, the animals were sacrificed and a wide rectangularincision was made in the skin of air sacs and photgraphed. Thesignificant stimulation of angiogenesis was detected in mice implantedwith B16BL6 melanoma cells. The treatment of animals with compound ofthis invention at dose 20 mg/kg/day inhibits tumor-induced angiogenesisto 70%.

Example 24

Alanine Substitution Mutations

The single point mutations in SEQ ID NO:1 insert coding sequence of thephage were produced as described previously (Hoess, R., Brinkmann, U.,Handel, T., Pastan, I. Gene, 128: 43–49, 1993.). Briefly, a series ofSEQ ID NO: 1 coding oligonucleotides in with a particular amino acidcoding triplet was changed to GCT (alanine coding triplet) weresynthesized. Mutated oligonucleotides were cloned in to fUSE5 phagevector as described in Example 16. All mutant phage clones were purifiedand verified by DNA sequencing.

The binding of mutated phage clones to immobilized Flt-1 receptor wasanalyzed by ELISA as described in Example 17.

TABLE 14 SEQ ID Binding to Clone Insert sequence NO. hFlt-1 in % V5.2NGYEIEWYSWVTHGMY 1 100(Asn-Gly-Tyr-Glu-Ile-Glu-Trp-Tyr-Ser-Trp-Val-Thr-His-Gly-Met-Tyr) N1/AAGYEIEWYSWVTHGMY 14 42.93 ± 25.76(Ala-Gly-Tyr-Glu-Ile-Glu-Trp-Tyr-Ser-Trp-Val-Thr-His-Gly-Met-Tyr) E4/ANGYAIEWYSWVTHGMY 15 17.54 ± 5.31 (Asn-Gly-Tyr-Ala-Ile-Glu-Trp-Tyr-Ser-Tr-Val-Thr-His-Gly-Met-Tyr) I5/ANGYEAEWYSWVTHGMY 16 18.76 ± 0.26 (Asn-Gly-Tyr-Glu-Ala-Glu-Trp-Tyr-Ser-Trp-Val-Thr-His-Gly-Met-Tyr) E6/ANGYEIAWYSWVTHGMY 17 19.155 ± 14.07 (Asn-Gly-Tyr-Glu-Ile-Ala-Trp-Tyr-Ser-Trp-Val-Thr-His-Gly-Met-Tyr) W7/ANGYEIEAYSWVTHGMY 18 66.23 ± 27.07(Asn-Gly-Tyr-Glu-Ile-Glu-Ala-Tyr-Ser-Trp-Val-Thr-His-Gly-Met-Tyr) Y8/ANGYEIEWASWVTHGMY 19 67.035 ± 6.28  (Asn-Gly-Tyr-Glu-Ile-Glu-Trp-Ala-Ser-Trp-Val-Thr-His-Gly-Met-Tyr) S9/ANGYEIEWYAWVTHGMY 20 101.69 ± 16.70 (Asn-Gly-Tyr-Glu-Ile-Glu-Trp-Tyr-Ala-Trp-Val-Thr-His-Gly-Met-Tyr) W10/ANGYEIEWYSAVTHGMY 21 22.08 ± 7.19 (Asn-Gly-Tyr-Glu-Ile-Glu-Trp-Tyr-Ser-Ala-Val-Thr-His-Gly-Met-Tyr) T12/ANGYEIEWYSWVAHGMY 22 62.63 ± 22.58(Asn-Gly-Tyr-Glu-Ile-Glu-Trp-Tyr-Ser-Trp-Val-Ala-His-Gly-Met-Tyr) H13/ANGYEIEWYSWVTAGMY 23 55.34 ± 10.78(Asn-Gly-Tyr-Glu-Ile-Glu-Trp-Tyr-Ser-Trp-Val-Thr-Ala-Gly-Met-Tyr) M15/ANGYEIEWYSWVTHGAY 24 78.24 ± 19.42(Asn-Gly-Tyr-Glu-Ile-Glu-Trp-Tyr-Ser-Trp-Val-Thr-His-Gly-Ala-Tyr) Y16/ANGYEIEWYSWVTHGMA 25 15.30 ± 15.27(Asn-Gly-Tyr-Glu-Ile-Glu-Trp-Tyr-Ser-Trp-Val-Thr-His-Gly-Met-Ala) E46/ANGYAIAWYSWVTHGMY 26 44.46 ± 7.05 (Asn-Gly-Tyr-Ala-Ile-Ala-Trp-Tyr-Ser-Trp-Val-Thr-His-Gly-Met-Tyr) W710/ANGYEIEAYSAVTHGMY 27 62.15 ± 22.16(Asn-Gly-Tyr-Glu-Ile-Glu-Ala-Tyr-Ser-Ala-Val-Thr-His-Gly-Met-Tyr) V5.2/1EIEWYSW 28  6.55 ± 15.81 (Glu-Ile-Glu-Trp-Tyr-Ser-Trp) V5.2/5EIEWYSWVTHGMY 29  85.6 ± 22.61(Glu-Ile-Glu-Trp-Tyr-Ser-Trp-Val-Thr-His-Gly-Met-Tyr)

Example 25

Association of SEQ ID NO: 1 with Erythropoietin.

The murine erythropoietin (mEPO) gene was cloned in pcDNA/Amp1.1expression vector using RT/PCR. CDNA encoded mEPO was obtained byreverse transcription of mRNA extracted from kidneys of mice treated for3 days with phenylhydrazine (Shoemaker C B., et al., Mol. Cel.Biol.,1986). Amplification of DNA was performed using the sense5′-ATAACAAGCTTGGCGCGGAGATGGGGGTG SEQ ID NO: 30 and antisense5′ATAACTCTAGAACGGTGGCAGCAGCATGTCAC SEQ ID NO: 31 primers. The amplifiedmEPO gene was inserted into the XbaI and Hind III sites of pcDNA/Amp1.1,and sequence was confirmed by DNA sequencing. Cos-7 cells weretransfected with pCMV/EPO plasmide, and expression of recombinant mEPOwas evaluated by Quantikine IVD Erythropoietin ELISA kit. Thephysiological activity of recombinant mEPO was evaluated in vivo bymeasured of hematocrit.

Synthetic oligonucleotide encoded peptide SEQ ID NO: 1 flanked withrestriction sites and SerGlyAlaGly linker (SEQ ID NO: 41) wassynthesized

(5′ACAACTCTAGAATGAACGGGTACGAGATCGAGTGGTACTCGTGGGTCACGCACGGGATGTACTCTGGGGCCGGATCTAGACAACA SEQ ID NO: 32).

Double strand DNA fragment was synthesized by using DNA extensionreaction and short complementary oligo. Further, the SEQ ID NO:1encoding DNA was restricted by Xba I and cloned in open reading frame ofmEPO into pCMV/EPO vector. Structure of SEQ ID NO:1-EPO fusion proteinwas confirmed by DNA sequencing.

Example 26

Construction of Adenoviral Vectors Encoding SEQ ID NO:1.

This example describes the construction of adenoviral vectors encodingfiber sequences having insertions of SEQ ID NO:1 peptide in a loop ofthe knob region of the adenovirus fiber protein.

The transfer plasmid p193 F5F2K(SP5.2) was employed to obtain thecorresponding adenoviral vectors comprising the SEQ ID NO: 1 peptide.This was accomplished by digesting these plasmids (which contain theessential E4 region of adenovirus) with Sal I, and transfecting theminto 293 cells that already had been infected 1 hour earlier with theadenovirus vector AdZ.E4Gus. This adenovirus vector lacks the E4 regionand cannot replicate in 293 cells without the E4 genes. Only whenAdZ.E4Gus DNA recombines with plasmid DNA such as p193 F5F2K, p193F5F2K(SP5.2) to obtain the E4 genes is the vector able to replicate in293 cells. During this recombination to rescue the adenoviral vector,the newly formed vector also picks up the mutated fiber sequence encodedby the plasmids.

Viable recombinant E4.sup.+adenovirus containing the F2K(SP5.2) DNAsequence (i.e., AdZ.SP5.2) was isolated by plaquing the transfected celllysates 5 days after transfection. The recombinant adenoviruses werethen plaque-purified 2 times on 293 cells. The purified plaques wereamplified on 293 cells. All viruses were purified from infected cells at2 days post-infection by 3 freeze-thaw cycles followed by two successivebandings on CsCl gradients. Purified virus was dialyzed into 10 mM Tris,150 mM NaCl, pH 7.8, containing 10 mM MgCl₂, 3% sucrose, and was frozenat −80 degree. until required for use. The purified viruses wereverified by PCR to contain SEQ ID NO: 1 encoding insert.

All publications or patents cited in this application are hereinincorporated by references.

1. A compound comprising amino acid sequenceGlu-Ile-Glu-Trp-Tyr-Ser-Trp-Val-Thr-His-Gly-Met-Tyr (SEQ ID NO:29).
 2. Apharmaceutical composition comprising the compound of claim 1 and abiological agent.
 3. A pharmaceutical composition comprising thecompound of claim 1 and a carrier.
 4. The compound of claim 1, whereinthe compound further comprises a biological agent conjugated thereto. 5.The compound of claim 4, wherein said biological agent comprises aprodrug.
 6. The compound of claim 4, wherein said biological agent is atherapeutic agent.